U.S. patent number 6,660,738 [Application Number 09/906,983] was granted by the patent office on 2003-12-09 for pyrrolidine derivatives.
This patent grant is currently assigned to Hoffmann-La Roche Inc.. Invention is credited to Johannes Aebi, Daniel Bur, Alexander Chucholowski, Henrietta Dehmlow.
United States Patent |
6,660,738 |
Aebi , et al. |
December 9, 2003 |
Pyrrolidine derivatives
Abstract
The present invention relates to pyrrolidine derivatives useful
as inhibitors of metalloproteases, e.g. zinc proteases, and which
are effective in treating disease states associated with
vasoconstriction.
Inventors: |
Aebi; Johannes (Basel,
CH), Bur; Daniel (Therwil, CH),
Chucholowski; Alexander (San Diego, CA), Dehmlow;
Henrietta (Grenzach-Wyhlen, DE) |
Assignee: |
Hoffmann-La Roche Inc. (Nutley,
NJ)
|
Family
ID: |
8169227 |
Appl.
No.: |
09/906,983 |
Filed: |
July 17, 2001 |
Foreign Application Priority Data
|
|
|
|
|
Jul 19, 2000 [EP] |
|
|
00114950 |
|
Current U.S.
Class: |
514/252.05;
514/252.02; 514/252.11; 514/255.05; 514/275; 514/343; 544/229;
544/238; 544/295; 544/296; 544/327; 544/331; 544/332; 544/336;
544/357; 546/256; 546/278.4 |
Current CPC
Class: |
A61P
9/12 (20180101); A61P 13/00 (20180101); C07D
401/04 (20130101); A61P 7/02 (20180101); A61P
15/08 (20180101); A61P 1/04 (20180101); A61P
43/00 (20180101); A61P 9/00 (20180101); A61P
3/10 (20180101); A61P 1/00 (20180101); A61P
17/02 (20180101); A61P 11/00 (20180101); A61P
31/04 (20180101); C07D 403/04 (20130101); A61P
9/06 (20180101); A61P 11/06 (20180101); A61P
9/04 (20180101); A61P 27/02 (20180101); A61P
25/08 (20180101); A61P 27/06 (20180101); A61P
13/12 (20180101); A61P 15/00 (20180101); A61P
9/10 (20180101); A61P 35/00 (20180101); A61P
37/06 (20180101); A61P 19/02 (20180101) |
Current International
Class: |
C07D
401/00 (20060101); C07D 403/00 (20060101); C07D
401/04 (20060101); C07D 403/04 (20060101); C07D
401/04 (); C07D 403/04 (); A61K 031/443 (); A61K
031/506 () |
Field of
Search: |
;544/229,238,327,331,332,336,295,296,357
;514/252.05,255.05,275,343,252.02,252.11 ;546/278.4,256 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
4962103 |
October 1990 |
Sunagawa et al. |
|
Foreign Patent Documents
|
|
|
|
|
|
|
0 411 664 |
|
Feb 1991 |
|
EP |
|
0 521 524 |
|
Jan 1993 |
|
EP |
|
0 528 678 |
|
Feb 1993 |
|
EP |
|
0 848 004 |
|
Jun 1998 |
|
EP |
|
WO 97/41120 |
|
Nov 1997 |
|
WO |
|
WO 98/08814 |
|
Mar 1998 |
|
WO |
|
WO 99/14195 |
|
Mar 1999 |
|
WO |
|
WO 99/52868 |
|
Oct 1999 |
|
WO |
|
Other References
Bertenshaw et al., PubMed Abstract (J. Biol. Chem. 278(4):2522-32),
Jan. 2003.* .
Yanagisawa, M. et al. Nature (Mar. 31, 1998), 332 (6/63), pp.
411-415. .
Xu, D., et al., Cell (1994) vol. 78 pp. 473-485. .
Oefner, et al., J. Mol. Biol. (2000) 296, pp. 341-349..
|
Primary Examiner: Rao; Deepak
Attorney, Agent or Firm: Johnston; George W. Parise; John
P.
Claims
What is claimed is:
1. A compound selected from the group consisting of compounds of
formula I ##STR8##
wherein R.sup.1 is hydrogen, alkylcarbonyl or arylcarbonyl; R.sup.2
is alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl,
alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl,
alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl,
aryl(alkoxycarbonyl)alkyl, arylaminocarbonyl, diarylalkyl,
aryl(carboxyalkyl)aminocarbonyl, arylcarbonyl, arylsulfonyl,
cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl,
heteroarylalkyl, heterocyclylalkyl or the group YR.sup.2 is
heterocyclyl; R.sup.3 and R.sup.4 are independently selected from
the group consisting of hydrogen, alkyl, alkylcycloalkyl,
alkylcycloalkylalkyl, alkylthio, cycloalkyl, cycloalkylalkyl,
carbamoyl, carboxy, carboxyalkyl, cyano, amino, mono- and
dialkylamino, alkoxy, alkoxyalkyl, alkoxycarbonyl,
alkoxycarbonylalkyl, alkenyl, alkinyl, aryl, arylalkyl,
arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, arylalkenyl,
aryl(alkoxycarbonyl)alkyl, arylamino, arylalkylamino, aryloxy,
halogen, heteroaryl, heteroarylalkyl, heterocyclyl,
heterocyclylalkyl, trimethylsilanylethynyl or trifluormethyl;
R.sup.5 is hydrogen, alkyl, aryl, arylalkyloxycarbonyl, or
alkylcarbonyl; X.sup.1, X.sup.2, X.sup.3 and X.sup.4 are CH or N
with the proviso that only up to two of X.sup.1, X.sup.2, X.sup.3
and X.sup.4 are N; and Y is --O-- or --NR.sup.5 --;
pharmaceutically acceptable esters and pharmaceutically acceptable
salts thereof.
2. The compound according to claim 1 of formula II ##STR9##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, X.sup.1, X.sup.2,
X.sup.3, X.sup.4 and Y are as defined in formula 1.
3. The compound according to claim 1 wherein R.sup.1 is hydrogen or
alkylcarbonyl.
4. The compound according to claim 3 wherein said alkylcarbonyl is
acetyl.
5. The compound according to claim 1 wherein R.sup.1 is
hydrogen.
6. The compound according to claim 1 wherein R.sup.2 is alkyl,
alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl,
alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl,
alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl,
aryl(alkoxycarbonyl)alkyl, diarylalkyl,
aryl(carboxyalkyl)aminocarbonyl, arylcarbonyl, arylsulfonyl,
cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl,
heteroarylalkyl, heterocyclylalkyl or arylaminocarbonyl which is
arylcarbamoyl.
7. The compound according to claim 1 wherein R.sup.2 is aryl,
arylalkyl, arylalkoxyalkyl, arylaminocarbonyl, arylcarbonyl,
arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl or
heteroarylalkyl.
8. The compound according to claim 1 wherein R.sup.2 is aryl,
arylalkyl, arylcarbonyl, arylsulfonyl, heteroarylalkyl, or
arylaminocarbonyl which is arylcarbamoyl.
9. The compound according to claim 8 wherein R.sup.2 is
arylalkyl.
10. The compound according to claim 9 wherein arylalkyl of R.sup.2
is phenylalkyl or phenylalkyl substituted with 2 or 3 halogen
atoms.
11. The compound according to claim 1 wherein R.sup.3 and R.sup.4
are independently selected from the group consisting of hydrogen,
alkyl, alkylthio, alkenyl, alkoxy, alkoxycarbonyl, amino, aryl,
arylalkyl, arylalkenyl, arylalkylamino, aryloxy, mono- and
dialkylamino, carbamoyl, carboxy, cyano, halogen, heteroaryl,
heteroarylalkyl, trimethylsilanylethynyl and trifluoromethyl.
12. The compound according to claim 1 wherein R.sup.3 and R.sup.4
are independently selected from the group consisting of hydrogen,
alkyl, alkoxy, alkoxycarbonyl, alkenyl, thiophenyl, amino, mono-
and dialkylamino, carboxy, cyano, halogen, trimethylsilanylethynyl,
phenylalkylamino, pyridinylpyrimidinyl, pyrazinyl, phenyl, and
phenoxy, wherein the aryl and heteroaryl groups of said thiophenyl,
phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl and
phenoxy are optionally substituted with alkyl, alkoxy, carboxy, or
halogen.
13. The compound according to claim 12 wherein R.sup.3 is hydrogen,
alkyl, alkoxy, alkoxycarbonyl, alkenyl, thiophenyl, amino, mono-
and dialkylamino, carboxy, cyano, halogen, trimethylsilanylethynyl,
phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl, and
phenoxy, wherein the aryl and heteroaryl groups of said thiophenyl,
phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl and
phenoxy are optionally substituted with alkyl, alkoxy, carboxy or
halogen, and wherein R.sup.4 is hydrogen.
14. The compound according to claim 1 wherein Y is --NR.sup.5
--.
15. The compound according to claim 1 wherein R.sup.5 is alkyl.
16. The compound according to claim 1 wherein R.sup.5 is
hydrogen.
17. The compound according to claim 1 wherein Y is --O--.
18. The compound according to claim 1 wherein X.sup.1 is N and
X.sup.2, X.sup.3 and X.sup.4 are each CH.
19. The compound according to claim 1 wherein X.sup.2 is N and
X.sup.1, X.sup.3 and X.sup.4 are each CH.
20. The compound according to claim 1 wherein X.sup.3 is N and
X.sup.1, X.sup.2 and X.sup.4 are each CH.
21. The compound according to claim 1 wherein X.sup.1, X.sup.2,
X.sup.3 and X.sup.4 are each CH.
22. The compound according to claim 1 wherein R.sup.1 is hydrogen
or alkylcarbonyl; R.sup.2 is arylalkyl which is phenylalkyl
substituted with 2 or 3 halogen atoms; R.sup.3 is hydrogen, alkyl,
alkoxy, alkoxycarbonyl, alkenyl, thiophenyl, amino, mono- and
dialkylamino, carboxy, cyano, halogen, trimethylsilanylethynyl,
phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl, phenyl, or
phenoxy, and wherein the aryl and heteroaryl groups of said
thiophenyl, phenylalkylamino, pyridinyl, pyrimidinyl, pyrazinyl,
phenyl and phenoxy are optionally substituted with alkyl, alkoxy,
carboxy, or halogen; R.sup.4 is hydrogen; X.sup.1, X.sup.2, X.sup.3
and X.sup.4 are CH or N with the proviso that only up to two groups
of X.sup.1, X.sup.2, X.sup.3 and X.sup.4 are N; and Y is --NH-- or
--O--.
23. The compound according to claim 22 wherein said alkylcarbonyl
of R.sup.1 is acetyl, and R.sup.2 is difluorobenzyl or
trifluorobenzyl.
24. The compound according to claim 1 selected from the group
consisting of: a)
(3R,5S)-1-pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3
-thiol trifluoro-acetate (1:1); b)
(3R,5S)-1-(4,6-dimethoxy-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; c)
(3R,5S)-1-(4-amino-5-fluoro-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxyme
thyl)-pyrrolidin-3-thiol; d)
2-[(2S,4R)-4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-nicotinonitrile; e)
(3R,5S)-1-(6-phenyl-pyridazin-3-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; f)
2-[(2S,4R)-4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-nicotinic acid; g)
2-[(2S,4R)-4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-6-methyl-pyrimidine-4-carboxylic acid methyl ester; h)
2-[(2S,4R)-4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-4-trifluoromethyl-pyrimidine-5-carboxylic acid methyl ester; i)
(3R,5S)-1-pyrazin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-t
hiol trifluoro-acetate; j)
2-[(2S,4R)-4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-nicotinamide; k)
(3R,5S)-5-(2,5-difluoro-4-methoxy-benzyloxymethyl)-1-(2-methoxy-pyrimidin-
4-yl)-pyrrolidine-3-thiol; l)
(3R,5S)-1-(2-chloro-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol trifluoro-acetate; m)
(3R,5S)-1-(5-ethyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol trifluoro-acetate (1:1); n)
(3R,5S)-1-(5-propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol trifluoro-acetate (1:1); o)
(3R,5S)-5-(2,4,5-trifluoro-benzyloxymethyl)-1-(4-trifluoromethyl-pyrimidin
-2-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1); p)
(3R,5S)-5-(2,4,5-trifluoro-benzyloxymethyl)-1-(5-trifluoromethyl-pyridin-2
-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1); q)
(3R,5S)-1-pyridin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-t
hiol trifluoro-acetate (1:1); r)
(2S,4R)-2-[4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-6-methyl-pyrimidine-4-carboxylic acid; s)
(3R,5S)-1-(2-methoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-p
yrrolidine-3-thiol; t)
(3R,5S)-1-(2-phenylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol trifluoro-acetate (1:1); u)
(3R,5S)-1-(2-benzylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; trifluoro-acetate (1:1); v)
(3R,5S)-1-(2-methylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; trifluoro-acetate (1:1); w) (3R,5S)
1-(2-butylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrol
idine-3-thiol; trifluoro-acetate (1:1); x)
(3R,5S)-1-(2-methylsulfanyl-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxyme
thyl)-pyrrolidine-3-thiol; y)
(3R,5S)-1-(2-phenoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-p
yrrolidine-3-thiol; z)
(3R,5S)-1-(5-phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; aa)
(3R,5S)-1-(5-pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid; bb)
(3R,5S)-1-(5-pyridin-4-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; cc)
(3R,5S)-1-(5-thiophen-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymet
hyl)-pyrrolidine-3-thiol; dd)
(3R,5S)-1-[5-(4-methoxy-phenyl)-pyrimidin-2-yl]-5-(2,4,5-trifluoro-benzylo
xymethyl)-pyrrolidine-3-thiol; ee)
(2S,4R)-4-{2-[4-mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-
yl]-pyrimidin-5-yl}-benzoic acid; ff)
(3R,5S)-1-(5-allyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol; gg)
(3R,5S)-1-(5-pyridin-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; and hh)
(3R,5S)-5-(2,4,5-trifluoro-benzyloxymethyl)-1-(5-trimethylsilanylethynyl-p
yrimidin-2-yl)-pyrrolidine-3-thiol.
25. The compound according to claim 1 selected from the group
consisting of: a)
(3R,5S)-1-pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3
-thiol trifluoro-acetate (1:1); b)
(3R,5S)-1-(6-phenyl-pyridazin-3-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; c)
(3R,5S)-1-pyrazin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-t
hiol; compound with trifluoro-acetic acid; d)
(3R,5S)-1-(5-ethyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol trifluoro-acetate (1:1); e)
(3R,5S)-1-(5-propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol trifluoro-acetate (1:1); f)
(3R,5S)-5-(2,4,5-trifluoro-benzyloxymethyl)-1-(5-trifluoromethyl-pyridin-2
-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1); g)
(3R,5S)-1-(5-phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; h) (3R,5S)-thioacetic acid
S-[1-(5-propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrodi
n-3-yl]ester; i)
(3R,5S)-1-(5-pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol trifluoro-acetate; j)
(3R,5S)-1-(5-pyridin-4-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; k)
1-(5-thiophen-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol; l)
1-(5-pyridin-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrr
olidine-3-thiol; m)
(2S,4R)-5-[(2,5-difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-
pyrrolidine-3-thiol; and n) (3R,5S)-thioacetic acid
S-[5-[(2,5-difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-pyrro
lidin-3-yl]ester.
26. A pharmaceutical composition comprising a compound according to
claim 1 and a pharmaceutically acceptable excipient.
27. A dimeric form of a compound of formula I ##STR10##
wherein R.sup.1 is hydrogen, alkylcarbonyl or arylcarbonyl; R.sup.2
is alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl,
alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl,
alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl,
aryl(alkoxycarbonyl)alkyl, arylaminocarbonyl, diarylalkyl,
aryl(carboxyalkyl)aminocarbonyl, arylcarbonyl, arylsulfonyl,
cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl,
heteroarylalkyl, heterocyclylalkyl or the group YR.sup.2 is
heterocyclyl; R.sup.3 and R.sup.4 are independently selected from
the group consisting of hydrogen, alkyl, alkylcycloalkyl,
alkylcycloalkylalkyl, alkylthio, cycloalkyl, cycloalkylalkyl,
carbamoyl, carboxy, carboxyalkyl, cyano, amino, mono- and
dialkylamino, alkoxy, alkoxyalkyl, alkoxycarbonyl,
alkoxycarbonylalkyl, alkenyl, alkinyl, aryl, arylalkyl,
arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, arylalkenyl,
aryl(alkoxycarbonyl)alkyl, arylamino, arylalkylamino, aryloxy,
halogen, heteroaryl, heteroarylalkyl, heterocyclyl,
heterocyclylalkyl, trimethylsilanylethynyl or trifluormethyl;
R.sup.5 is hydrogen, alkyl, aryl, arylalkyloxycarbonyl, or
alkylcarbonyl; X.sup.1, X.sup.2, X.sup.3 and X.sup.4 are CH or N
with the proviso that only up to two of X.sup.1, X.sup.2, X.sup.3
and X.sup.4 are N; and Y is --O-- or --NR.sup.5 --.
28. A pharmaceutical composition comprising a dimeric form of a
compound according to claim 27 and a pharmaceutically acceptable
excipient.
Description
BACKGROUND OF THE INVENTION
Endothelins are peptides, that exist in three isoforms ET-1, ET-2,
and ET-3, each encoded by a distinct gene. They have been
originally discovered in the conditioned medium of porcine
endothelial cells in 1988 by Yanagisawa (Yanagisawa M; Kurihara H;
Kimura S; Tomobe Y; Kobayashi M; Mitsui Y; Yazaki Y; Goto K; Masaki
T: A novel potent vasoconstrictor peptide produced by vascular
endothelial cells [see comments]. NATURE (Mar. 31, 1988),
332(6163), 411-5.). The active ETs are peptides of 21 amino acids
with two intramolecular disulfide bridges. They are produced from
preproproteins of 203 to 212 amino acids which are processed by
furin-like endopeptidases to the biologically inactive
big-endothelin (big-ET). The big-ETs are specifically processed to
mature ETs by a hydrolytic cleavage between amino acids 21 and 22
that are Trp.sup.21 -Val.sup.22 (big-ET-1, big ET-2) and Trp.sup.21
-Ile.sup.22 in big-ET-3 respectively. Already in 1988 a specific
metalloprotease was postulated to be responsible for this specific
cleavage. In 1994, ECE-1 (endothelin converting enzyme-1) was
purified and cloned from bovine adrenal (Xu D, Emoto N, Giaid A,
Slaughter C, Kaw S, de Witt D, Yanagisawa M: ECE-1: a
membrane-bound metalloprotease that catalyzes the proteolytic
activation of big endothelin-1. Cell (1994) 78: 473-485).
ECE-1 is a membrane bound type II zinc-endopeptidase with a neutral
pH optimum and a zinc binding motif HExxHx(>20)E. It belongs to
subfamily M13 and has a large 681 amino acid ectodomain that
comprises the active site. Other members of the M13 family are
NEP24.11 (neutral endopeptidase), PEX, a phosphate regulating
neutral endopeptidase, and Kell blood group protein that has
recently been described as a big-ET-3 processing enzyme. Members of
the M13 family of human origin are characterized by a high
molecular weight (>80 kDa) a number of conserved disulfide
bridges and a complex glycosylation pattern. The structure of NEP
has recently been solved. (Oefner et al, J. Mol. Biol. 2000, 296,
341-349). The catalytic domain of ECE and related human M13
proteinases are significantly larger (>650 amino acids) than
members of matrix metalloproteases (MMPs). Unlike the family of the
MMPs which belong to the metzincins and display a typical
HExxHxxGxxH pattern members of the M13 family are gluzincins
comprising a HExxHx(>20)E pattern. These two families are
clearly different in size of catalytic domains, structure and zinc
coordinating pattern of ligands. Active sites of the two families
show clear differences which has clear impact on type of inhibitors
and the potential selectivity.
SUMMARY OF THE INVENTION
The present invention relates to compounds of formula (I)
##STR1##
wherein R.sup.1 is hydrogen, alkylcarbonyl or arylcarbonyl; R.sup.2
is alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl,
alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl,
alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl,
aryl(alkoxycarbonyl)alkyl, arylaminocarbonyl, diarylalkyl,
aryl(carboxyalkyl) aminocarbonyl, arylcarbonyl, arylsulfonyl,
cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl,
heteroarylalkyl, heterocyclylalkyl or the group YR.sup.2 is
heterocyclyl; R.sup.3 and R.sup.4 are independently selected from
the group consisting of hydrogen, alkyl, alkylcycloalkyl,
alkylcycloalkylalkyl, alkylthio, cycloalkyl, cycloalkylalkyl,
carbamoyl, carboxy, carboxyalkyl, cyano, amino, mono- and
dialkylamino, alkoxy, alkoxyalkyl, alkoxycarbonyl,
alkoxycarbonylalkyl, alkenyl, alkinyl, aryl, arylalkyl,
arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, arylalkenyl,
aryl(alkoxycarbonyl)alkyl, arylamino, arylalkylamino, aryloxy,
halogen, heteroaryl, heteroarylalkyl, heterocyclyl,
heterocyclylalkyl, trimethylsilanylethynyl or trifluormethyl;
R.sup.5 is hydrogen, alkyl, aryl, arylalkyloxycarbonyl, or
alkylcarbonyl; X.sup.1, X.sup.2, X.sup.3 and X.sup.4 are CH or N
with the proviso that only up to two groups of X.sup.1, X.sup.2,
X.sup.3 and X.sup.4 are N; Y is --O-- or --NR.sup.5 --; and dimeric
forms, and/or pharmaceutically acceptable esters, and/or
pharmaceutically acceptable salts thereof, preferably
pharmaceutically acceptable esters, and/or pharmaceutically
acceptable salts thereof, and most preferably pharmaceutically
acceptable salts thereof.
The present invention is directed to compounds which are useful as
inhibitors of metalloproteases, e.g. zinc proteases, particularly
zinc hydrolases, and which are effective in the prophylaxis and
treatment of disease states which are associated with
vasoconstriction of increasing occurrences. Examples of such
disorders are high blood pressure, coronary disorders, cardiac
insufficiency, renal and myocardial ischaemia, renal insufficiency,
dialysis, cerebral ischaemia, cardiac infarct, migraine,
subarachnoid haemorrhage, Raynaud syndrome and pulmonary high
pressure. In addition the compounds are useful as cytostatic and
cerebroprotective agents for inhibition of graft rejection, for
organ protection and for treatment of ophthalmological
diseases.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to compounds of formula (I):
##STR2##
wherein R.sup.1 is hydrogen, alkylcarbonyl or arylcarbonyl; R.sup.2
is alkyl, alkinyl, hydroxyalkyl, carboxyalkyl, alkoxycarbonyl,
alkylcarbonylalkyl, alkylcycloalkyl, alkylcycloalkylalkyl,
alkylsulfonyl, aryl, arylalkyl, arylalkoxyalkyl,
aryl(alkoxycarbonyl)alkyl, arylaminocarbonyl, diarylalkyl,
aryl(carboxyalkyl)aminocarbonyl, arylcarbonyl, arylsulfonyl,
cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl, heteroaryl,
heteroarylalkyl, heterocyclylalkyl or the group YR.sup.2 is
heterocyclyl; R.sup.3 and R.sup.4 are independently selected from
the group consisting of hydrogen, alkyl, alkylcycloalkyl,
alkylcycloalkylalkyl, alkylthio, cycloalkyl, cycloalkylalkyl,
carbamoyl, carboxy, carboxyalkyl, cyano, amino, mono- and
dialkylamino, alkoxy, alkoxyalkyl, alkoxycarbonyl,
alkoxycarbonylalkyl, alkenyl, alkinyl, aryl, arylalkyl,
arylalkyl(alkoxycarbonyl)alkyl, arylcarbonylalkyl, arylalkenyl,
aryl(alkoxycarbonyl)alkyl, arylamino, arylalkylamino, aryloxy,
halogen, heteroaryl, heteroarylalkyl, heterocyclyl,
heterocyclylalkyl, trimethylsilanylethynyl or trifluormethyl;
R.sup.5 is hydrogen, alkyl, aryl, arylalkyloxycarbonyl, or
alkylcarbonyl; X.sup.1, X.sup.2, X.sup.3 and X.sup.4 are CH or N
with the proviso that only up to two groups of X.sup.1, X.sup.2,
x.sup.3 and X.sup.4 are N; Y is --O-- or --NR.sup.5 --; and dimeric
forms, and/or pharmaceutically acceptable esters, and/or
pharmaceutically acceptable salts thereof.
The term "alkyl", alone or in combination, means a straight-chain
or branched-chain alkyl group containing a maximum of 7, preferably
a maximum of 4, carbon atoms, e.g., methyl, ethyl, n-propyl,
2-methylpropyl (iso-butyl), 1-methylethyl (iso-propyl), n-butyl,
and 1,1-dimethylethyl (t-butyl).
The term "carboxy" refers to the group --C(O)OH.
The term "carbamoyl" refers to the group --C(O)NH.sub.2.
The term "carbonyl" refers to the group --C(O)--.
The term "halogen" refers to the group fluoro, bromo, chloro and
iodo.
The term "sulfonyl" refers to the group --S(O.sub.2)--.
The term "alkenyl" refers to a hydrocarbon chain as defined for
alkyl having at least one olefinic double bond (including for
example, vinyl, allyl and butenyl).
The term "alkinyl" refers to a hydrocarbon chain as defined for
alkyl having at least one olefinic triple bond (including for
example propinyl, butin-(1)-yl, etc.).
The term "alkoxy", alone or in combination, means an alkyl ether
group in which the term `alkyl` has the significance given earlier,
such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy,
isobutoxy, sec.butoxy, tert.butoxy and the like.
The term "alkoxycarbonyl" refers to a group of the formula
--C(O)R.sub.c wherein R.sub.c is alkoxy as defined above.
The term "hydroxy" refers to the group --OH, the term "cyano" to
the group --CN.
The term "hydroxyalkyl" means an alkyl group as defined above which
is substituted by a hydroxy group.
The term "thioalkyl" and "cyanoalkyl" refer to an alkyl group as
defined above which is substituted by a --SH group or an --CN
group, respectively.
"Carboxyalkyl" means a lower-alkyl as defined above which is
substituted by a HOOC-- group.
The term "alkylcarbonyl", alone or in combination, means an acyl
group derived from an alkanecarboxylic acid, i.e. alkyl--C(O)--,
such as acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl
etc.
The term "cycloalkyl" signifies a saturated, cyclic hydrocarbon
group with 3-8, preferably 3-6 carbon atoms, i.e. cyclopropyl,
cyclobutyl, cyclopentyl and cyclohexyl and the like.
The term "amino" refers to the group --NH.sub.2.
The term "aryl" for R.sup.2 -- alone or in combination--, refers to
an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic
or partially aromatic ring, e.g. phenyl, naphthyl or
tetrahydronaphthyl, preferably phenyl or naphthyl, and most
preferably phenyl. The aryl moiety is optionally substituted with
one or more groups independently selected from halogen, preferably
fluor, alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl,
alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy,
1,3-dioxolyl, or 1,4-dioxolyl, more preferably fluor,
alkoxycarbonyl, alkyl, trifluoromethyl and trifluoromethoxy and
most preferably fluor. The most preferred aromatic groups are
2,5-difluorobenzyl and 2,4,5-trifluorobenzyl.
The term "aryl" for R.sup.3 and R.sup.4 --alone or in
combination--, refers to an aromatic carbocyclic radical, i.e. a 6
or 10 membered aromatic or partially aromatic ring, e.g. phenyl,
naphthyl or tetrahydronaphthyl, preferably phenyl or naphthyl, and
most preferably phenyl. The aryl moiety is optionally substituted
with one or more groups independently selected from halogen,
alkoxycarbonyl, e.g. methylcarbonyl, carboxy, cyano, alkyl, alkoxy,
phenyl, phenoxy, trifluormethyl, trifluormethoxy, 1,3-dioxolyl, or
1,4-dioxolyl, cyclohexyl, hydroxy, alkylamido, e.g. acetamido,
nitro, alkylsulfonyl, e.g. methylsulfonyl, more preferably fluor,
chlor, brom, alkoxy, carboxy, 1,4-dioxolyl, alkoxycarbonyl. The
most preferred aromatic groups for R.sup.3 and R.sup.4 are phenyl
and phenoxy.
The term "aryl" for R.sup.5 refers to phenyl optionally substituted
with alkyl, alkoxy or halogen.
The term "aryloxy" refers to an aryl group as defined above
attached to a parent structure via an oxy radical, i.e.,
aryl-O--.
The term "heteroaryl" for R.sup.2 --alone or in combination--refers
to an aromatic mono- or bicyclic radical having 5 to 10, preferably
5 to 6 ring atoms, containing one to three heteroatoms, preferably
one heteroatom, e.g. independently selected from nitrogen, oxygen
or sulfur. Examples of heteroaryl groups are thiophenyl,
isoxazolyl, thiazolyl, pyridinyl, pyrrolyl, imidazolyl, tetrazolyl,
preferably pyridinyl, isoxazolyl and thiazolyl. Optionally, the
heteroaryl group can be mono-, di- or tri-substituted,
independently, with phenyl, alkyl, alkylcarbonyl, alkoxycarbonyl,
hydroxy, amino, alkylamino, dialkylamino, carboxy,
alkoxycarbonylalkyl, preferably alkyl.
The term "heteroaryl" for R.sup.3 and R.sup.4 --alone or in
combination--refers to an aromatic mono- or bicyclic radical having
5 to 10, preferably 5 to 6 ring atoms, containing one to three
heteroatoms, preferably one heteroatom, e.g. independently selected
from nitrogen, oxygen or sulfur. Examples of heteroaryl groups are
pyridinyl, thiophenyl, isoxyzolyl, isoquinolyl, quinolyl, and
1H-benzo[d][1,3]oxazin-2,4-dione and indolyl, pyrimidine,
pyridazine, and pyrazine, preferably pyridinyl and thiophenyl.
Optionally, the heteroaryl group can be mono, di- or
tri-substituted, independently, with alkyl, alkoxy, halogen,
alkylcarbonyl, alkoxycarbonyl, hydroxy, amino, alkylamino,
dialkylamino, carboxy, alkoxycarbonylalkyl, preferably alkyl.
The term "heterocyclyl" --alone or in combination--refers to a
non-aromatic mono- or bicyclic radical having 5 to 10, preferably 5
to 6 ring atoms, containing one to three heteroatoms, preferably
one heteroatom, e.g. independently selected from nitrogen, oxygen
or sulfur. Optionally the heterocyclic ring can be substituted by a
group independently selected from halogen, alkyl, alkoxy,
oxocarboxy, alkoxycarbonyl, etc. and/or on a secondary nitrogen
atom (i.e. --NH--) by alkyl, arylalkoxycarbonyl, alkylcarbonyl or
on a tertiary nitrogen atom (i.e. .dbd.N--) by oxido. Examples for
heterocyclic groups are morpholinyl, pyrrolidinyl, piperidyl,
etc.
The term "dimeric form" means a compound wherein the two R.sup.1
groups of two identical compounds of formula I have been replaced
by a common single bond or wherein R.sup.1 is glutathione-S-- or
cysteine-S-- or ester and/or alkylcarbonyl or arylcarbonyl
derivatives thereof, e.g. acetylcysteine-S-- or
benzoylcysteine-S--, preferably glutathione-S--, cysteine-S--,
acetylcysteine-S-- or benzoylcysteine-S--.
The term "pharmaceutically acceptable salt" refers to those salts
which retain the biological effectiveness and properties of the
free bases or free acids, which are not biologically or otherwise
undesirable. The salts are formed with inorganic acids such as
hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic
acid, propionic acid, glycolic acid, pyruvic acid, oxylic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid, N-acetylcysteine and the like. In addition these
salts may be prepared from addition of an inorganic base or an
organic base to the free acid. Salts derived from an inorganic base
include, but are not limited to, the sodium, potassium, lithium,
ammonium, calcium, magnesium salts and the like. Salts derived from
organic bases include, but are not limited to salts of primary,
secondary, and tertiary amines, substituted amines including
naturally occurring substituted amines, cyclic amines and basic ion
exchange resins, such as isopropylamine, trimethylamine,
diethylamine, triethylamine, tripropylamine, ethanolamine, lysine,
arginine, N-ethylpiperidine, piperidine, polymine resins and the
like.
"Pharmaceutically acceptable esters" means that compounds of
general formula (I) may be derivatised at functional groups to
provide derivatives which are capable of conversion back to the
parent compounds in vivo. Examples of such compounds include
physiologically acceptable and metabolically labile ester
derivatives, such as methoxymethyl esters, methylthiomethyl esters
and pivaloyloxymethyl esters. Additionally, any physiologically
acceptable equivalents of the compounds of general formula (I),
similar to the metabolically labile esters, which are capable of
producing the parent compounds of general formula (I) in vivo, are
within the scope of this invention.
The compounds of formula (I) are useful in inhibiting mammalian
metalloprotease activity, particularly zinc hydrolase activity.
More specifically, the compounds of formula (I) are useful as
medicaments for the treatment and prophylaxis of disorders which
are associated with diseases caused by endothelin-converting enzyme
(ECE) activity. Inhibiting of this enzyme would be useful for
treating myocardial ischaemia, congestive heart failure,
arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral
vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney
diseases, atherosclerosis, Buerger's disease, Takayasu's arthritis,
diabetic complications, lung cancer, prostatic cancer,
gastrointestinal disorders, endotoxic shock and septicaemia, and
for wound healing and control of menstruation, glaucoma. In
addition the compounds are useful as cytostatic and
cerebroprotective agents, for inhibition of graft rejection, for
organ protection and for treatment of ophthalmological
diseases.
A preferred embodiment of the present invention encompasses
compounds of general formula (II) ##STR3##
wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, X.sup.1, X.sup.2,
X.sup.3, X.sup.4 and Y are as defined in formula 1.
In a preferred embodiment of the invention R.sup.1 is selected from
hydrogen or alkylcarbonyl, more preferably from hydrogen or acetyl
and most preferably R.sup.1 is hydrogen.
In the above compounds R.sup.2 is preferably alkyl, alkinyl,
hydroxyalkyl, carboxyalkyl, alkoxycarbonyl, alkylcarbonylalkyl,
alkylcycloalkyl, alkylcycloalkylalkyl, alkylsulfonyl, aryl,
arylalkyl, arylalkoxyalkyl, aryl(alkoxycarbonyl)alkyl,
arylcarbamoyl, diarylalkyl, aryl(carboxyalkyl)amide, arylcarbonyl,
arylsulfonyl, cycloalkyl, cycloalkylcarbonyl, cycloalkylalkyl,
heteroaryl, heteroarylalkyl or heterocyclylalkyl, more preferably
R.sup.2 is aryl, arylalkyl, arylalkoxyalkyl, arylaminocarbonyl,
arylcarbonyl, arylsulfonyl, cycloalkyl, cycloalkylcarbonyl,
cycloalkylalkyl or heteroarylalkyl, even more preferably R.sup.2 is
aryl, arylalkyl, arylcarbamoyl, arylamino, arylcarbonyl,
arylsulfonyl or heteroarylalkyl and most preferably R.sup.2 is
arylalkyl. In an especially preferred embodiment of the present
invention R.sup.2 is phenylalkyl optionally substituted with 2 to 3
halogen atoms, preferably fluor atoms.
In the compounds of the present invention, preferably R.sup.3 and
R.sup.4 are independently selected from the group consisting of
hydrogen, alkyl, alkylthio, alkenyl, alkoxy, alkoxycarbonyl, amino,
aryl, arylalkyl, arylalkenyl, arylalkylamino, aryloxy, mono- and
dialkylamino, carbamoyl, carboxy, cyano, halogen, heteroaryl,
heteroarylalkyl, trimethylsilanylethynyl and trifluoromethyl, more
preferably R.sup.3 and R.sup.4 are independently selected from the
group consisting of hydrogen, alkyl, alkoxy, alkoxycarbonyl,
alkenyl, thiophenyl, amino, mono- and dialkylamino, carboxy, cyano,
halogen, trimethylsilanylethynyl, phenylalkylamino, pyridinyl,
pyrimidinyl, pyrazinyl, phenyl, and phenoxy, wherein the aryl and
heteroaryl groups are optionally substituted with alkyl, alkoxy,
carboxy, or halogen. In the most preferred embodiment of the
present invention R.sup.3 is hydrogen, alkyl, alkoxy,
alkoxycarbonyl, alkenyl, thiophenyl, amino, mono- and dialkylamino,
carboxy, cyano, halogen, trimethylsilanylethynyl, phenylalkylamino,
pyridinyl, pyrimidinyl, pyrazinyl, phenyl, and phenoxy, wherein the
aryl and heteroaryl groups are optionally substituted with alkyl,
alkoxy, carboxy, or halogen, and R.sup.4 is hydrogen.
In a further preferred embodiment of the present invention Y is
--NR.sup.5 --R.sup.5 being hydrogen or alkyl and more preferably
hydrogen.
In another preferred embodiment of the present invention Y is
--O--.
The invention also relates to the above defined compounds wherein
X.sup.1 is N and X.sup.2, X.sup.3 and X.sup.4 are CH, or wherein
X.sup.2 is N and X.sup.1, X.sup.3 and X.sup.4 are CH or wherein
X.sup.3 is N and X.sup.1, X.sup.2 and X.sup.4 are CH or wherein
X.sup.1, X.sup.2, X.sup.3 and X.sup.4 are CH.
A preferred embodiment the present invention comprises compounds as
defined above wherein R.sup.1 is hydrogen or alkylcarbonyl, R.sup.2
is phenylalkyl substituted with 2 to 3 halogen; R.sup.3 is selected
from hydrogen, alkyl, alkoxy, alkoxycarbonyl, alkenyl, thiophenyl,
amino, mono- and dialkylamino, carboxy, cyano, halogen,
trimethylsilanylethynyl, phenylalkylamino, pyridinyl, pyrimidinyl,
pyrazinyl, phenyl, or phenoxy, and wherein the aryl and heteroaryl
groups are optionally substituted with alkyl, alkoxy, carboxy, or
halogen; R.sup.4 is hydrogen; X.sup.1, X.sup.2, X.sup.3 and X.sup.4
are CH or N with the proviso that only up to two groups of X.sup.1,
X.sup.2, X.sup.3 and X.sup.4 are N; and Y is --NH-- or --O--. In a
preferred embodiment R.sup.1 is hydrogen or acetyl and R.sup.2 is
difluorobenzyl or trifluorobenzyl in the above defined
compounds.
Preferred embodiments of the present invention are the compounds
exemplified in the examples. Especially the present invention
comprises compounds selected from the group consisting of a)
(3R,5S)-1-Pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3
-thiol trifluoro-acetate (1:1); b)
(3R,5S)-1-(4,6-Dimethoxy-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; c)
(3R,5S)-1-(4-Amino-5-fluoro-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxyme
thyl)-pyrrolidine-3-thiol; d)
2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-nicotinonitrile; e)
(3R,5S)-1-(6-Phenyl-pyridazin-3-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; f)
2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-nicotinic acid; g)
2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-6-methyl-pyrimidine-4-carboxylic acid methyl ester; h)
2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-4-trifluoromethyl-pyrimidine-5-carboxylic acid methyl ester; i)
(3R,5S)-1-Pyrazin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-t
hiol; compound with trifluoro-acetic acid; j)
2-[(2S,4R)-4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-nicotinamide; k)
(3R,5S)-5-(2,5-Difluoro-4-methoxy-benzyloxymethyl)-1-(2-methoxy-pyrimidin-
4-yl)-pyrrolidine-3-thiol; l)
(3R,5S)-1-(2-Chloro-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; compound with trifluoro-acetic acid; m)
(3R,5S)-1-(5-Ethyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol trifluoro-acetate (1:1); n)
(3R,5S)-1-(5-Propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol trifluoro-acetate (1:1); o)
(3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(4-trifluoromethyl-pyrimidin
-2-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1); p)
(3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trifluoromethyl-pyridin-2
-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1); q) (3R,5S
)-1-Pyridin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-thiol
trifluoro-acetate (1:1); r)
(2S,4R)-2-[4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-6-methyl-pyrimidine-4-carboxylic acid; s)
(3R,5S)-1-(2-Methoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-p
yrrolidine-3-thiol; t)
(3R,5S)-1-(2-Phenylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol trifluoro-acetate (1:1); u)
(3R,5S)-1-(2-Benzylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; trifluoro-acetate (1:1); v)
(3R,5S)-1-(2-Methylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; trifluoro-acetate (1:1); w)
(3R,5S)-1-(2-Butylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl
)-pyrrolidine-3-thiol; trifluoro-acetate (1:1); x)
(3R,5S)-1-(2-Methylsulfanyl-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxyme
thyl)-pyrrolidine-3-thiol; y)
(3R,5S)-1-(2-Phenoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-p
yrrolidine-3-thiol; z)
(3R,5S)-1-(5-Phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; aa)
(3R,5S)-1-(5-Pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid; bb)
(3R,5S)-1-(5-Pyridin-4-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; cc)
(3R,5S)-1-(5-Thiophen-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymet
hyl)-pyrrolidine-3-thiol; dd)
(3R,5S)-1-[5-(4-Methoxy-phenyl)-pyrimidin-2-yl]-5-(2,4,5-trifluoro-benzylo
xymethyl)-pyrrolidine-3-thiol; ee)
(2S,4R)-4-{2-[4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-
yl]-pyrimidin-5-yl}-benzoic acid; ff)
(3R,5S)-1-(5-Allyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol; gg)
(3R,5S)-1-(5-Pyridin-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; and hh)
(3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trimethylsilanylethynyl-p
yrimidin-2-yl)-pyrrolidine-3-thiol.
These compounds show IC.sub.50 values in the radioimmunoassay (E on
ECE-inhibition, see blow) of about 0.5 nM to 100 nM.
Especially preferred compounds as defined by formula (I) are those
selected from the group consisting of a)
(3R,5S)-1-Pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3
-thiol trifluoro-acetate (1:1); b)
(3R,5S)-1-(6-Phenyl-pyridazin-3-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; c)
(3R,5S)-1-Pyrazin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3-t
hiol; compound with trifluoro-acetic acid; d)
(3R,5S)-1-(5-Ethyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol trifluoro-acetate (1:1); e)
(3R,5S)-1-(5-Propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol trifluoro-acetate (1:1); f)
(3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trifluoromethyl-pyridin-2
-yl)-pyrrolidine-3-thiol trifluoro-acetate (1:1); g)
(3R,5S)-1-(5-Phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol; h) (3R,5S)-Thioacetic acid
S-[1-(5-propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrroli
din-3-yl]ester; i)
(3R,5S)-1-(5-Pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid; j)
(3R,5S)-1-(5-Pyridin-4-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; k)
1-(5-Thiophen-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol; l)
1-(5-Pyridin-3-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrr
olidine-3-thiol; m)
(2S,4R)-5-[(2,5-Difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-
pyrrolidine-3-thiol; and n) (3R,5S)-Thioacetic acid
S-[5-[(2,5-difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-pyrro
lidin-3-yl]ester.
The invention also refers to pharmaceutical compositions containing
a compound as defined above and a pharmaceutically acceptable
excipient.
A further embodiment of the present invention refers to the use of
compounds as defined above as active ingredients in the manufacture
of medicaments comprising a compound as defined above for the
prophylaxis and treatment of disorders which are caused by
endothelin-converting enzyme (ECE) activity especially myocardial
ischaemia, congestive heart failure, arrhythmia, hypertension,
pulmonary hypertension, asthma, cerebral vasospasm, subarachnoid
haemorrhage, pre-eclampsia, kidney diseases, atherosclerosis,
Buerger's disease, Takayasu's arthritis, diabetic complications,
lung cancer, prostatic cancer, gastrointestinal disorders,
endotoxic shock and septicaemia, and for wound healing and control
of menstruation, glaucoma, graft rejection, diseases associated
with cytostatic, ophthalmological, and cerebroprotective
indications, and organ protection.
Further the invention refers to the use of compounds as described
above for the treatment or prophylaxis of diseases which are
associated with myocardial ischaemia, congestive heart failure,
arrhythmia, hypertension, pulmonary hypertension, asthma, cerebral
vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney
diseases, atherosclerosis, Buerger's disease, Takayasu's arthritis,
diabetic complications, lung cancer, prostatic cancer,
gastrointestinal disorders, endotoxic shock and septicaemia, and
for wound healing and control of menstruation, glaucoma, graft
rejection, diseases associated with cytostatic, ophthalmological,
and cerebroprotective indications, and organ protection.
In addition the invention comprises compounds as described above
for use as therapeutic active substances, in particular in context
with diseases which are associated with zinc hydrolase activity
such as myocardial ischaemia, congestive heart failure, arrhythmia,
hypertension, pulmonary hypertension, asthma, cerebral vasospasm,
subarachnoid haemorrhage, pre-eclampsia, kidney diseases,
atherosclerosis, Buerger's disease, Takayasu's arthritis, diabetic
complications, lung cancer, prostatic cancer, gastrointestinal
disorders, endotoxic shock and septicaemia, and for wound healing
and control of menstruation, glaucoma, graft rejection, diseases
associated with cytostatic, ophthalmological, and cerebroprotective
indications, and organ protection.
The invention also comprises a method for the therapeutic and/or
prophylactic treatment of myocardial ischaemia, congestive heart
failure, arrhythmia, hypertension, pulmonary hypertension, asthma,
cerebral vasospasm, subarachnoid haemorrhage, pre-eclampsia, kidney
diseases, atherosclerosis, Buerger's disease, Takayasu's arthritis,
diabetic complications, lung cancer, prostatic cancer,
gastrointestinal disorders, endotoxic shock and septicaemia, and
for wound healing and control of menstruation, glaucoma, graft
rejection, diseases associated with cytostatic, ophthalmological,
and cerebroprotective indications, and organ protection, which
method comprises administering a compound as defined above to a
human being or animal.
The invention also relates to the use of compounds as defined above
for the inhibition of zinc hydrolase activity.
The invention also refers to the above compounds whenever
manufactured by a process as described below.
Compounds of formula (I) can be prepared by methods known in the
art or as described below. Unless otherwise indicated, the
substituents R.sup.1, R.sup.2, R.sup.3 R.sup.4, R.sup.5, X.sup.1,
X.sup.2, X.sup.3,X.sup.4, and Y mentioned below are as defined
above.
The process for the preparation of a compound as defined above may
comprise the reaction of a compound of formula III ##STR4##
wherein A is a HS- and P is a NH-protecting group as described in
the following sections, a) with a R.sup.2 -halogenide for
introduction of a --OR.sup.2 group followed by P-deprotection and
introduction of a heteroaromate: or b) first P-deprotection of
formula (III), introduction of a heteroaromate as defined above
followed by --OH/--NH.sub.2 replacement and reductive amination to
introduce R.sup.2 ;
optionally followed by conversion of a R.sup.1, R.sup.2, R.sup.3,
R.sup.4 group as above into a different one and/or deprotection
and/or thiol liberation.
For the preparation of compounds of formula (I) the reaction
pathway of scheme 1 can be followed: the starting material is
commercial available or is synthesized from hydroxyproline by
methods known in the art and described for example in "The Practice
of Peptide Synthesis", M. Bodanszky and A. Bodanszky, Springer
Verlag, Berlin, 1984.
The synthesis starts with the inversion of the configuration via
preparation of the corresponding mesylate (e.g. reaction with
MeSO.sub.3 H/Ph.sub.3 P/DIAD in toluene at RT to 80.degree. C.),
via the chloride (e.g. reaction with Ph.sub.3 P/CCl.sub.4 in
CH.sub.2 Cl.sub.2 at 3.degree. C. to RT) or via the bromide (e.g.
reaction with LiBr/DEAD/Ph.sub.3 P in THF at 4.degree. C. to RT).
For retention of configuration the corresponding reaction may be
performed with MeSO.sub.2 Cl/pyridine/DMAP at 0.degree. C. to
RT.
Step b of scheme 1 shows the introduction of the protected thio
moiety, e.g. by reaction with triphenylmethanthiol or
4-methoxybenzylmercaptane (K-Ot-Bu in DMF for Cl: 0.degree. C., for
Br: 0.degree. C. to RT, for Mes: RT to 100.degree. C.).
Reaction of step c of scheme 1 may be performed via Method A (LAH
in THF at -20.degree. C.) or Method B (Red-Al in toluene/THF at
-50.degree. C.).
Reaction of step d (for Y.dbd.--O--) may be performed with 1.
NaH/R.sup.2 Br in DMF 0.degree. C. to RT, (O-alkylation) 2. TFA in
CH.sub.2 Cl.sub.2 -20 to RT, (BOC deprotection) 3. Method A:
2-Chloro-hetero-aromate/N-ethyldiisopropylamine 3 h 80.degree. C.,
Method B (parallel-synthesis):
2-Chloro-hetero-aromate/N-ethyldiisopropylamine in dioxane or DMF,
16 h -2days 80-130.degree. C.,Method C (for less reactive
compounds): 2-Chloro-hetero-aromate/N-ethyldiisopropylamine/CuI 10
h 80.degree. C.
For the preparation of phenolether compounds, the corresponding
reaction may be performed under Mitsunobu conditions (DEAD/Ph.sub.3
P/PhOH or PhSH in THF).
For Y being NR.sup.2 or N-heterocycle a mesylation reaction may be
performed: e.g. 1. 1.1 eq MeSO.sub.2 Cl/1.5pyridine/1 eq DMAP,
(mesylation); 2. YR.sup.2 is e.g. pyrrole, imidazol or, 1 eq NaI,
NaH in DMF 0.degree. C. to RT; 3. iPr.sub.3 SiH in TFA/CH.sub.2
Cl.sub.2 or CH.sub.3 CN (for trityl-thiol deprotection).
Thiol liberation may be performed with TFA/iPr.sub.3 SiH in
CH.sub.2 Cl.sub.2 or CH.sub.3 CN at RT.
An alternative route for the preparation of compounds with Y being
N is: first P-deprotection (TFA in CH.sub.2 Cl.sub.2 at -20.degree.
C. to RT for P.dbd.BOC), followed by reaction with
2-chloro-hetero-aromate/N-ethyldiisopropylamine/CuI for 10 h at
80.degree. C. (step f) followed by 1. phthalimide, DEAD/Ph.sub.3 P
in THF 0 to 80.degree. C., (phthalimide introduction under
Mitsunobu condition) 2. hydrazine hydrate, EtOH, RT, (phthalimide
deprotection) followed by reaction with the corresponding 3.
aldehyde, SnCl.sub.2, NaBH.sub.3 CN, MeOH, (reductive amination)
(step g) 4. If necessary. R.sup.5 may be introduced by reaction
with R.sup.5 Br/K.sub.2 CO.sub.3 in acetonitrile, RT, followed by
reaction with 5. iPr.sub.3 SiH in TFA/CH.sub.2 Cl.sub.2 or CH.sub.3
CN (for trityl-thiol deprotection). ##STR5##
Scheme 2 summarizes special reaction pathways for the preparation
of compounds of formula (I):
Scheme 2 A refers to a Cl-derivative as starting material which was
synthesized as described in Scheme 1 (step e) with
2,4-dichloropyrimidine: 1. If XR.sup.3 is OMe the reaction may be
performed with 3 eq MeOH/NaH in DMF, RT (4-fluoro-replacement takes
place in YR.sup.2.dbd.2,4,5-trifluoro benzyloxy ether derivatives).
2. If XR.sup.3 is OPh, the reaction may be performed with 10 eq
PhOH/NaH in DMF for 8 h at 70.degree. C. 3. If XR.sup.3 is OMe, the
reaction may be performed with 2.2 eq MeONa in MeOH at RT to
75.degree. C. (10 h) (no 4-fluoro-replacement takes place in
YR.sup.2.dbd.2,4,5-trifluoro-benzyloxy ether derivatives). 4. If
XR.sup.3 is SMe, the reaction may be performed with 2.2 eq
MeSNa/Nal in THF, RT to 70.degree. C. for 28 h. 5. If XR.sup.3 is
NHR.sup.3, the reaction may be performed with 7.5-30 eq H.sub.2
NR.sup.3 /iPr.sub.2 EtN in dioxane at 90-105.degree. C. for 48 h.
6. Thiol liberation may then be performed with TFA/iPr.sub.3 SiH in
CH.sub.2 Cl.sub.2 or CH.sub.3 CN at RT (step b).
The reaction pathway of scheme 2 B shows further synthesis routes
for compounds of formula (I):
Reaction of step c) may be performed with the following methods: 1.
Suzuki-coupling with ArylB(OH).sub.2 /Pd(PhP).sub.4 in
dimethoxyethane/EtOH and 2 M Na.sub.2 CO.sub.3 2 h at 90.degree.
C.; or 2. reaction with ArylB(OH).sub.2 /PdCl.sub.2 (dppf) in
dioxane and 2 M Na.sub.2 CO.sub.3 for 24-48 h at 80.degree. C.; or
3. i. synthesis of a boron ester (e.g.
4,4,5,5-Tetramethyl-2-phenyl-[1,3,2]dioxaborolane derivative) by
reaction with bis(pinacolato)diboron/KOAc/PdCl.sub.2 (dppf) in DMF
at 80.degree. C., and 4. ii. reaction with Bromoaromat/PdCl.sub.2
(dppf)/2 M Na.sub.2 CO.sub.3 for 16 h at 80.degree. C.; or
Sonogashira-Hagihara coupling: reaction with
ethinyltrimethylsilane/Et.sub.3 N/PdCl.sub.2 (Ph.sub.3 P)2/CuI in
DMF at 80.degree. C. ##STR6## ##STR7##
Further derivatization of compounds of formula (I) is described in
Scheme 3:
In case Y is nitrogen, reaction of step a may be performed with
RCOCl, iPr.sub.2 NEt, 4-(N-Benzyl-N-methylamino)pyridine
polymer-supported, CH.sub.2 Cl.sub.2 (N-acylation) followed by
reaction with iPr.sub.3 SiH, TFA, CH.sub.2 Cl.sub.2, (thiol
liberation). In case Y is protected nitrogen or oxygen, the
reaction with the free thiol may be performed according to step c
with RCOCl in pyridine at 0.degree. C. to RT or BOC-Cys(Npys)-OH
(=2-(BOC-Cys)disulfanyl-3-nitro-pyridine) in DMF/0.1 M phosphate
buffer (pH 6.2). In case of Y is a benzyloxy protected nitrogen,
selective deprotection with 33% HBr in acetic acid at 0.degree. C.
to RT is possible.
Dimeric forms of a compound of formula I may be prepared by
oxidative treatment of the formula I monomers.
On the basis of their capability of inhibiting metalloprotease
activity, especially zinc hydrolase activity, the compounds of
formula I can be used as medicaments for the treatment and
prophylaxis of disorders which are associated with vasoconstriction
of increasing occurrences. Examples of such disorders are high
blood pressure, coronary disorders, cardiac insufficiency, renal
and myocardial ischaemia, renal insufficiency, dialysis, cerebral
ischaemia, cardiac infarct, migraine, subarachnoid haemorrhage,
Raynaud syndrome and pulmonary high pressure. They can also be used
in atherosclerosis, the prevention of restenosis after
balloon-induced vascular dilation, inflammations, gastric and
duodenal ulcers, ulcus cruris, gram-negative sepsis, shock,
glomerulonephtritis, renal colic, glaucoma, asthma, in the therapy
and prophylaxis of diabetic complications and complications in the
administration of cyclosporin, as well as other disorders
associated with endothelin activities.
The ability of the compounds of formula (I) to inhibit
metalloprotease activity, particularly zinc hydrolase activity,
maybe demonstrated by a variety of in vitro and in vivo assays
known to those of ordinary skill in the art. Pharmaceutically
acceptable esters, pharmaceutically acceptable salts and dimeric
forms of the compounds of formula I can also be tested by those of
ordinary skill in the art for their ability to inhibit
metalloprotease activity.
A) Cell Culture
A stable human umbilical vein endothelial cell line (ECV304) was
cultured in "cell factories" as described until confluency
(Schweizer et al. 1997, Biochem. J. 328: 871-878). At confluency
cells were detached with a trypsin/EDTA solution and collected by
low speed centrifugation. The cell pellet was washed once with
phosphate buffered saline pH 7.0 and stored at -80.degree. C. until
use.
B) Solubilization of ECE from ECV304 Cells
All procedures were performed at 0-4.degree. C. if not stated
otherwise. The cell pellet of 1.times.10.sup.9 cells was suspended
in 50 ml of buffer A (20 mM Tris/HCl, pH 7.5 containing 5 mM
MgCl.sub.2, 100 .mu.M PMSF, 20 .mu.M E64, 20 .mu.M leupeptin) and
sonicated. The resulting cell homogenate was centrifuged at 100,000
g.sub.av for 60 minutes. The supernatant was discarded and the
resulting membrane pellet was homogenized in 50 ml buffer A and
centrifugated as described. The washing of the membrane fraction in
buffer A was repeated twice. The final membrane preparation was
homogenized in 50 ml of buffer B (buffer A+0.5% Tween 20 (v/v),
0.5% CHAPS (w/v), 0.5% Digitonin (w/v)) and stirred at 4.degree. C.
for 2 hours. Thereafter the remaining membrane fragments were
sedimented as described. The resulting clear supernatant containing
the solubilized ECE was stored in 1.0 ml aliquots at -120.degree.
C. until use.
C) ECE Assay
The assay measured the production of ET-1 from human big ET-1. To
measure high numbers of samples an assay performed in 96 well
plates was invented. The enzyme reaction and the radioimmunological
detection of the produced ET-1 was performed in the same well,
using a specifically developed and optimized coating technique.
D) Coating of Plates
Fluoronunc Maxisorp White (code 437796) 96 well plates were
irradiated with 1 joule for 30 minutes in a UV Stratalinker 2400
(Stratagene). The 96 well plates were then fill with 300 .mu.l
protein A solution (2 .mu.g/ml in 0.1 M Na.sub.2 CO.sub.3 pH 9.5)
per well and incubated for 48 hours at 4.degree. C. Coated plates
can be stored for up to 3 weeks at 4.degree. C. until use.
Before use the protein A solution is discarded and the plates are
blocked for 2 hours at 4.degree. C. with 0.5% BSA in 0.1M Na.sub.2
CO.sub.3, pH 9.5.
Plates were washed with bidestilled water and were ready to perform
the ECE assay.
E) Screening Assay
Test compounds are solved and diluted in DMSO. 10 .mu.l of DMSO was
placed in the wells, followed by 125 .mu.l of assay buffer (50 mM
Tris/HCl, pH 7.0, 1 .mu.M Thiorphan, 0,1% NaN.sub.3, 0.1% BSA)
containing 200 ng big ET-1. The enzyme reaction was started by the
addition of 50 .mu.l of solubilized ECE (diluted in assay buffer
1:30 to 1:60 fold (v/v)). The enzyme reaction was carried out for
30 minutes at 37.degree. C. The enzyme reaction was stopped by
addition of 10 .mu.l 150 mM ETDA, pH 7.0.
Radioimmunoassay
The ET-1 RIA was performed principally as described earlier
(Loffler, B.-M. and Maire, J.-P. 1994, Endothelium 1: 273-286). To
plates containing the EDTA stopped enzyme reaction mixture 25 .mu.l
of assay buffer containing 20000 cpm (3-(.sup.125
I)Tyr)-endothelin-1 and 25 .mu.l of the ET specific antiserum AS-3
(dilution in assay buffer 1:1000) was added. Plates were incubated
under mixing at 4.degree. C. over night. Thereafter, the liquid
phase was sucked with a plate washer and plates were washed once
with bidestilled water. To the washed plates 200 .mu.l
scintillation cocktail (Microscint 40 LSC-Cocktail, Packard, code
6013641) was added and plates were counted for 2 minutes per well
in a Topcount.
Standard curves were prepared in plates with synthetic ET-1 with
final concentrations of 0 to 3000 pg ET-1 per well. In all plates
controls for maximal ECE activity (in the presence of 10 .mu.l
DMSO) and for background production of ET-1 immunoreactivity (in
the presence of 10 mM EDTA or 100 .mu.M phosphoramidon) were
performed. Assays were run in triplicate.
F) Kinetic Assay
The described assay format could be used to determine the kinetic
characteristics of the used ECE preparation as well as different
ECE inhibitors (i.e. Km, Ki) by variation of the substrate
concentration used in the assay.
G) Cell based ECE Assay
Human ECE-1c was stable expressed in MDCK cells as described
(Schweizer et al. 1997, Biochem. J. 328: 871-878). Cells were
cultured in 24 well plates to confluency in Dulbecco's modified
Eagles's medium (DMEM) supplemented with 10% (v/v) fetal bovine
serum (FBS), 0.8 mg/ml geneticin, 100 i.u./ml penicillin and 100
.mu.g/ml streptomycin in a humidified air/CO.sub.2 (19:1)
atmosphere. Before ECE assay the medium was replaced by 0.5 ml
DMEM-HBSS 1:1, 10 mM HEPES pH 7.0 supplemented with 0.1% (w/v) BSA.
The inhibitors were added in DMSO at a final concentration of 1%.
The enzyme reaction was started by the addition of 0.42 .mu.M human
big ET-1 and performed for 1.5 hours at 37.degree. C. in an
incubator. At the end of incubation, the incubation medium was
quickly removed and aliquots were analysed by radioimmunoassay for
produced ET-1 as described above.
The ECE screening assay was validated by the measurement of the
characteristic inhibitor constants of phosphoramidon (IC.sub.50
0.8.+-.0.2 .mu.M) and CGS 314447 (IC.sub.50 20.+-.4 nM) [De
Lombaert, Stephane; Stamford, Lisa B.; Blanchard, Louis; Tan,
Jenny; Hoyer, Denton; Diefenbacher, Clive G.; Wei, Dongchu;
Wallace, Eli M.; Moskal, Michael A.; et al. Potent non-peptidic
dual inhibitors of endothelin-converting enzyme and neutral
endopeptidase 24.11. Bioorg. Med. Chem. Lett. (1997), 7(8),
1059-1064]. The two inhibitors were measured with IC.sub.50 values
not significantly different from those described in the literature
but measured with different assay protocols. In the cell based
assay phosphoramidon showed an IC.sub.50 of 4 .mu.M. This assay
gave additional information about the inhibitory potency of
inhibitors under much more physiologic conditions, as e.g. the ECE
was embedded in a normal plasma membrane environment. It is
important to state, that the screening assay was performed in the
presence of 1 .mu.M Thiorphan to block any potential big ET-1
degradation due to the action of NEP24.11. No NEP activity was
present in MDCK-ECE-1c transfected cells in preliminary experiments
when ET-1 production was measured in presence or absence of
thiorphan. In subsequent experiments no thiorphan was added in the
incubation medium.
According to the above methods, the compounds of the present
invention show IC.sub.50 values in the radioimmunoassay (E on
ECE-inhibition) of about 0.5 nM to about 100 .mu.M. The preferred
compounds show values of 0.5 nM to 100 nM.
As mentioned earlier, medicaments containing a compound of formula
I are also an object of the present invention as is a process for
the manufacture of such medicaments, which process comprises
bringing one or more compounds of formula I and, if desired, one or
more other therapeutically valuable substances into a galenical
administration form.
The pharmaceutical compositions may be administered orally, for
example in the form of tablets, coated tablets, dragees, hard or
soft gelatin capsules, solutions, emulsions or suspensions.
Administration can also be carried out rectally, for example using
suppositories; locally or percutaneously, for example using
ointments, creams, gels or solutions; or parenterally, for example
using injectable solutions.
For the preparation of tablets, coated tablets, dragees or hard
gelatin capsules the compounds of the present invention may be
admixed with pharmaceutically inert, inorganic or organic
excipients. Examples of suitable excipients for tablets, dragees or
hard gelatin capsules include lactose, maize starch or derivatives
thereof, talc or stearic acid or salts thereof.
Suitable excipients for use with soft gelatin capsules include for
example vegetable oils, waxes, fats, semi-solid or liquid polyols
etc.; according to the nature of the active ingredients it may
however be the case that no excipient is needed at all for soft
gelatin capsules.
For the preparation of solutions and syrups, excipients which may
be used include for example water, polyols, saccharose, invert
sugar and glucose. For injectable solutions, excipients which may
be used include for example water, alcohols, polyols, glycerin, and
vegetable oils. For suppositories, and local or percutaneous
application, excipients which may be used include for example
natural or hardened oils, waxes, fats and semi-solid or liquid
polyols.
The pharmaceutical compositions may also contain preserving agents,
antioxidants, solubilising agents, stabilizing agents, wetting
agents, emulsifiers, sweeteners, colorants, odorants, salts for the
variation of osmotic pressure, buffers, coating agents or
antioxidants. They may also contain other therapeutically valuable
agents.
The dosages in which the compounds of formula I are administered in
effective amounts depend on the nature of the specific active
ingredient, the age and the requirements of the patient and the
mode of application. In general, dosages of 0.1-100 mg/kg body
weight per day come into consideration, although the upper limit
quoted can be exceeded when this is shown to be indicated.
The following specific examples are provided as a guide to assist
in the practice of the invention, and are not intended as a
limitation on the scope of the invention.
EXAMPLES
All reactions are done under argon.
A) Abbreviations
EtOAc ethylacetate, EtOH ethanol, THF tetrahydrofurane, Et.sub.2 O
diethylether, MeOH methanol, CH.sub.2 Cl.sub.2 dichloromethane, DMF
dimethylformamide, BOC t-butyloxycarbonyl, LAH Lithium aluminium
hydride, LDA lithium diisopropylamide, DEAD Diethyl
azodicarboxylate, DIAD Diisopropyl azodicarboxylate, DMAP
4-Dimethylamino-pyridine, iPr.sub.2 NEt N-ethyldiisopropylamine,
Ph.sub.3 P triphenylphosphine, Red-Al solution
Natrium-dihydrido-bis-(2-methoxyethoxy)-aluminat-solution, Et.sub.3
N triethylamine, ArylB(OH).sub.2.dbd.aryl-, heteroaryl-,
alpha-alkenyl boronic acid, PdCl.sub.2
(dppf)(1,1'-bis(diphenylphosphino)ferrocene)dichloropalladium
(II).CH.sub.2 Cl.sub.2 (1:1), Pd(Ph.sub.3 P).sub.4
tetrakis(triphenylphosphine) palladium, iPr.sub.3 SiH
triisopropylsilane, PdCl.sub.2 (Ph.sub.3 P).sub.2
bis(triphenylphosphine) palladium(II) dichloride, Et.sub.3 SiH
triethylsilane, TFA trifluoroacetic acid.
B) General Method for a Selective BOC-deprotection
A solution of 15.1 mmol N-BOC-S-Trityl compound in 30 ml CH.sub.2
Cl.sub.2 was treated at -20.degree. C. with 34 ml TFA and warmed up
to room temperature during 5.5 h. The reaction was evaporated and
treated with aqueous saturated NaHCO.sub.3 solution/EtOAc
(3.times.) to give the free aminotritylsulfanyl.
C) General Method for Ester Hydrolysis
A solution of 5.38 mmol carboxylic acid methyl ester was dissolved
in 150 ml EtOH and treated at RT with 10.8 ml (10.8 mmol) aqueous 1
N NaOH. After 3 h the reaction was evaporated and poured into
aqueous 10% KHSO.sub.4 /EtOAc (3.times.). The organic phases were
washed with aqueous 10% NaCl solution and dried over Na.sub.2
SO.sub.4 to give the carboxylic acid.
D) General Method for S-deprotection
Trityl Deprotection with Triisopropylsilane: A solution of 2.84
mmol trityl-protected compound in 30 ml CH.sub.2 Cl.sub.2 was
treated at 0.degree. C. with 8 ml TFA and 5.82 ml (28 mmol)
triisopropylsilane. After 30 min at RT the solution was completely
evaporated and the compound precipitated twice from Et.sub.2
O/pentane or purified by silcagel with cyclohexane,
cyclohexane/EtOAc 9:1 to 1:1 as eluent to give the thiol
trifluoro-acetate (1:1) as colourless oil.
E) General Method for S-deprotection Modified
1 eq tritylated educt in CH.sub.2 Cl.sub.2 (20 ml/mmol) was treated
with 10-20 eq triisopropyl silane and 10-20 eq TFA at 0.degree. C.
The solution was stirred at 0.degree. C. until no educt was
detected, was poured on sat. NaHCO.sub.3 solution and was extracted
with CH.sub.2 Cl.sub.2. The combined organic phases were washed
with brine and are dried over Na.sub.2 SO.sub.4.
Example 1
Starting Materials
The starting material:
(2S,4R)-4-tritylsulfanyl-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester for the synthesis of the compound
of the present invention are known in the art and described for
example in International Patent Application WO98/20001 and European
Patent Application Publication No. EP-A-696593.
1.1 (2S,4S)-4-Chloro-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl
ester 2-methyl ester
A solution of 374 g (1.48 mol)
(2S,4R)-4-Hydroxy-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl
ester 2-methyl ester in 1.6 l CH.sub.2 Cl.sub.2 was treated with
680 g (2.6 mol) triphenylphosphine, cooled to 3-5.degree. C. and
treated in 10 min with 1.241 (12.8 mol) CCl.sub.4, after 2 h at
this temperature cooling was stopped, the reaction raised during 2
h to 35.degree. C. It was cooled down to 20.degree. C. and stirred
for further 45 min. After addition of 4 l of n-heptane, the
reaction was evaporated to 2.9 l, cooled to 0.degree. C., filtered,
the residue was treated twice the same way, the third time by
dissolving the residue again in 2 l of CH.sub.2 Cl.sub.2. The
solvents were evaporated and filtered through silica gel with
hexane/tert.-butyl-methylether 9:1 as eluent. Evaporation of the
solvents gave 347 g (89%) of
(2S,4S)-4-Chloro-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl
ester 2-methyl ester, MS: 246 (MH.sup.+).
1.2 (2S,4R)-4-Tritylsulfanyl-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester
A solution of 76 g (0.68 mol) potassium-tert.-butylate in 1.5 l DMF
was cooled (-3.degree. C.) and treated slowly (1.5 h) with 202 g
(0.73 mol) triphenylmethanethiol in 0.8 l DMF (at max 1.degree.
C.). After 2.5 h at 0.degree. C., a solution of 161 g (0.61 mol) of
(2S,4S)-4-Chloro-pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl
ester 2-methyl ester in 0.35 l DMF was added. The reaction was
stirred over night at 2.degree. C., evaporated, dissolved in 1.5 l
EtOAc, poured into 2.7 l aqueous saturated NH.sub.4 Cl solution and
extracted with EtOAc (2.times.). The organic phase was washed with
aqueous saturated NaHCO.sub.3, dried over Na.sub.2 SO.sub.4 and
evaporated. Colum chromatographyon silica gel with hexane/EtOAc
(95:5 to 7:3) gave 268 g (87%)
(2S,4R)-4-Tritylsulfanyl-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester, MS: 504 (MH.sup.30 ).
1.3 Ester Reduction
A)
(2S,4R)-2-Hydroxymethyl-4-tritylsulfanyl-pyrrolidine-1-carboxylic
acid Tert-butyl ester
A solution of 35 g (69 mmol)
(2S,4R)-4-Tritylsulfanyl-pyrrolidine-1,2-dicarboxylic acid
1-tert-butyl ester 2-methyl ester in 380 ml toluene/60 ml THF was
treated at -47.degree. C. to -50.degree. C. with 44 g (152 mmol) of
a 70% solution of sodium dihydrido-bis(2-methoxy-ethoxo)aluminate
in toluene (3.5 M Red-Al in toluene). After 3 h at -50.degree. C.
and 1 h at -30.degree. C. the solution was poured into water (1 l)
with 40 g of citric acid and extracted with EtOAc (2.times.). The
organic phase was dried over Na.sub.2 SO.sub.4 and evaporated.
Column chromatography on silica gel with hexane/EtOAc (7:3) gave
23.0 g (69%)
(2S,4R)-2-Hydroxymethyl-4-tritylsulfanyl-pyrrolidine-1-carboxylic
acid tert-butyl ester, MS: 476 (MH+).
B)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine-1
-carboxylic acid Tert-butyl ester
A solution of 15.5 g (32.59 mmol)
(2S,4R)-2-Hydroxymethyl-4-tritylsulfanyl-pyrrolidine-1-carboxylic
acid tert-butyl ester and 24.7 g (109.77 mmol)
2,4,5-trifluoro-benzylbromide in 700 ml DMF at 0.degree. C. was
treated with 2.28 g (52.14 mmol) of 55% NaH in 4 portions and
warmed up to RT during 7 h. The reaction was cooled to 0.degree. C.
and treated with 500 ml aqueous saturated NH.sub.4 Cl solution,
extracted with EtOAc (3.times.). The organic phase was washed with
10% NaCl dried over Na.sub.2 SO.sub.4 and evaporated. Flash column
chromatography on silica gel with hexane/EtOAc (9:1 to 8.5:1.5)
gave 9.37 g (46%) of
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine-1
-carboxylic acid tert-butyl ester, MS: 620 (MH.sup.+).
C)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine
A solution of 9.37 g (15.11 mmol)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine-1
-carboxylic acid tert-butyl ester in 30 ml CH.sub.2 Cl.sub.2 was
treated at -20.degree. C. with 34 ml TFA and warmed up to RT during
5.5 h. The reaction was evaporated and treated with aqueous sat
NaHCO.sub.3 solution/EtOAc (3.times.) to give 7.77 g (quantitative)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine,
MS: 520 (M).
Example 2
N-Pyrrolidine Derivatives (Scheme 2)
2.1 Method A
A mixture of 2.08 g (4
mmol)(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolid
ine, 0.687 g (6 mmol) 2-chloropyrimidine and 1.16 ml (6.8 mmol)
N-ethyldiisopropylamine was heated for 3 h at 80.degree. C. The
reaction was cooled and partitioned between H.sub.2 O/Et.sub.2 O
(3.times.300). The organic phases were washed with aqueous
saturated NaHCO.sub.3, aqueous 10% NaCl, dried (NaSO.sub.4) and
evaporated. Flash chromatography on silica gel (CH.sub.2 Cl.sub.2
/EtOAc 97.5:2.5) gave 1.7 g (71%)
(2S,4R)-2-[2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidin
-1-yl]-pyrimidine, MS: 598 (MH.sup.+).
In analogy: a)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine
and 2,4-dichloropyrimidine gave
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine, MS: 632 (MH.sup.+); b)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine
and 2,5-dibromo-primidine [Brown, Desmond J.; Arantz, B. W.,
Pyrimidine reactions. XXII. Relative reactivities of corresponding
chloro-, bromo-, and iodopyrimidines in aminolysis. J. Chem. Soc. C
(1971), Issue 10, 1889-91] gave
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine, MS: 676 (MH.sup.+, 1Br); c)
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine
and methyl-2-chloro-6-methylpyrimidine gave
(2S,4R)-6-Methyl-2-[2-(2,4,5-trifluoro-benzyloxy-methyl)-4-tritylsulfanyl-
pyrrolidin-1-yl]-pyrimidine-4-carboxylic acid methyl ester, MS: 670
(MH.sup.+); d)
(2S,4R)-6-Methyl-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine-4-carboxylic acid methyl ester was
hydrolyzed following the general method for hydrolysis of an ester
(ETOH/dioxane) to give
(2S,4R)-6-Methyl-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine-4-carboxylic acid, MS: 656
(MH.sup.+).
2.2 S-Deprotection, Method D)
A solution of 1.7 g (2.84 mmol)
(2S,4R)-2-[2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidin
-1-yl]-pyrimidine in 30 ml CH.sub.2 Cl.sub.2 was treated at
0.degree. C. with 8 ml TFA and 5.82 ml (28 mmol)
triisopropylsilane. After 30 min at RT the solution was completely
evaporated and the compound precipitated twice from Et.sub.2
O/pentane to give 1.06 g (80%)
(3R,5S)-1-pyrimidin-2-yl-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidine-3
-thiol trifluoro-acetate (1:1) as colourless oil, MS: 356
(MH.sup.+).
In analogy: a)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine gave (3R,5S)
1-(2-Chloro-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin
e-3-thiol; compound with trifluoro-acetic acid, MS: 390 (MH.sup.+);
b)
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine gave
(2S,4R)-1-(5-Bromo-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyr
rolidine-3-thiol, MS: 434 (MH.sup.+,1Br). (nicht in Liste vorne);
c)
(2S,4R)-6-Methyl-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine-4-carboxylic acid gave
(2S,4R)-2-[4-Mercapto-2-(2,4,5-trifluoro-benzyloxymethyl)-pyrrolidin-1-yl]
-6-methyl-pyrimidine-4-carboxylic acid, MS: 414 (MH.sup.+).
2.3 Method B (Synthesis in Parallel)
A solution of 0.45 mmol
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine
in 1 ml dioxane or 0.1 ml DMF, 2.25 mmol of 2-chloro-hetero-aromate
and 2.25 mmol N-ethyldiisopropylamine was heated for 16 h-2 days at
80-130.degree. C. (see table 1). The reaction was purified by
preparative HPLC (RP-18, MeCN/H2O, UV 230 nm).
TFA/triisopropylsilane deprotection as described (see General
method for S-deprotection, Method D) gave the free thiol.
2.4 Method C
A mixture of 2 g (3.85
mmol)(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolid
ine, 1.2 g (7.7 mmol) 2-chloro5-n-propylpyrimidine, 1.98 ml (11.55
mmol) N-ethyldiisopropylamine and a catalytic amount of copper(I)
iodide was heated for 10 h at 80.degree. C. The reaction was cooled
and partitioned between H.sub.2 O/Et.sub.2 O (3.times.300). The
organic phases were washed with aqueous saturated NaHCO.sub.3,
aqueous 10% NaCl, dried (NaSO.sub.4) and evaporated. Flash
chromatography on silica gel (toluene/Et.sub.2 O 99:1) gave 2 g
(81%)
(2S,4R)-5-Propyl-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine, MS: 640 (MH.sup.+).
TFA/triisopropylsilane deprotection as described (see General
method for S-deprotection, Method D) gave
(3R,5S)-1-(5-Propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol trifluoro-acetate (1:1), MS: 398 (MH.sup.+).
According to an analogous method the following compounds were
prepared via reaction of
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidine
with the 2.educt mentioned in the following table 1.
TABLE 1 By the reaction of
(2S,4R)-2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-
pyrrolidine 66-7030 with the 2.educt. Solvent/ Time/Te NAME 2.
Educt Method mp./.degree. C. MS (3R,5S)-1-(4,6-Dimethoxy-
2-CHLORO-2,4- B DMF cat 416 M+H+
pyrimidin-2-yl)-5-(2,4,5-trifluoro- DIMETHOXY- KI/16 h/
benzyloxymethyl)-pyrrolidine-3- PYRIMIDINE 80 thiol
(3R,5S)-1-(4-Amino-5-fluoro- 4-AMINO-2- B DMF/24 389 M+H+
pyrimidin-2-yl)-5-(2,4,5-trifluoro- CHLORO-5- h/120
benzyloxy-methyl)-pyrrolidine-3- FLUORO- thiol PYRIMIDINE
2-[(2S,4R)-4-Mercapto-2-(2,4,5- METHYL 2-CHLORO- B no/16 h/ 482
M+H+ trifluoro-benzyloxy-methyl)- 4-(TRIFLUORO- 80
pyrrolidin-1-yl]-4-trifluoromethyl- METHYL) pyrimidine-5-carboxylic
acid PYRIMIDINE-5- methyl ester CARBOXYLATE
2-[(2S,4R)-4-Mercapto-2-(2,4,5- METHYL 2-CHLORO- B no/16 h/ 428
M+H+ trifluoro-benzyloxy-methyl)- 6-METHYL- 80
pyrrolidin-1-yl]-6-methyl- PYRIMIDINE-4- pyrimidine-4-carboxylic
acid CARBOXYLATE methyl ester (3R,5S)-1-(5-Ethyl- 2-CHLORO-5- C
no/10 h/ 384 M+H+ pyrimidin-2-yl)-5-(2,4,5- ETHYL- 80
trifluoro-benzyloxy- PYRIMIDINE methyl)-pyrrolidine-3-thiol
trifluoro-acetate (1:1) (3R,5S)-5-(2,4,5- 2-CHLORO-4- B no/16 h/
424 M+H+ Trifluoro-benzyloxy- (TRIFLUOROMETHYL) 85
methyl)-1-(4-trifluoro- PY-RIMIDINE methyl-pyrimidin-2-yl)-
pyrrolidine-3-thiol trifluoro-acetate (1:1)
2-[(2S,4R)-4-Mercapto-2- 2-CHLORO- B no/16 h/ 380 M+H+
(2,4,5-trifluoro- NICOTINO- 80 benzyloxymethyl)- NITRILE
pyrrolidin-1-yl]- nicotinonitrile 2-[(2S,4R)-4-Mercapto- 2-CHLORO-
B dioxane, 399 M+H+ 2-(2,4,5-trifluoro- NICOTINIC ACID DMF/24 h/
benzyloxymethyl)- 120 pyrrolidin-1-yl]-nicotinic acid
2-[(2S,4R)-4-Mercapto- 2-CHLORO- B dioxane/16 398 M+H+
2-(2,4,5-trifluoro- NICOTIN-AMIDE h/80 benzyloxymethyl)-
pyrrolidin-1-yl]- nicotinamide (3R,5S)-5-(2,4,5- 2-CHLORO-5- B
no/16 h/ 423 M+H+ Trifluoro-benzyloxy- (TRIFLUORO 80
methyl)-1-(5-trifluoro- METHYL) methyl-pyridin-2-yl)- PYRIDINE
pyrrolidine-3-thiol trifluoro-acetate(1:1) (3R,5S)-1-Pyridin-2-yl-
2-CHLORO- B no/2 days/ 355 M+H+ 5-(2,4,5-trifluoro- PYRIDINE 130
benzyloxymethyl)- pyrrolidine-3-thiol trifluoro-acetate(1:1)
(3R,5S)-1-Pyrazin-2-yl- 2-CHLORO- C no/45 min/ 470 M+H+
5-(2,4,5-trifluoro- PYRAZINE 160 benzyloxymethyl)-
pyrrolidine-3-thiol; compound with trifluoro-acetic acid
(3R,5S)-1-(6-Phenyl- 3-CHLORO-6- B DMF/16 h/ 432 M+H+
pyridazin-3-yl)-5-(2,4,5- PHENYL- 80 trifluoro-benzyloxy PYRIDAZINE
methyl)-pyrrolidine-3- thiol
Example 3
Substitution on 2-Chloropyrimidine
3.1 Reaction in DMF
A solution of 0.24 (0.4 mmol)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine in 8 ml DMF was treated at 0.degree. C.
with 0.05 ml (1.2 mmol) MeOH and 0.054 g (1.24 mmol) 55% NaH. The
reaction was kept at this temperature (6 h) and warmed up over
night to RT. After extraction with aqueous saturated NH.sub.4
Cl/Et.sub.2 O (3.times.), the organic phases were washed with aq.
10% NaCl, dried (Na.sub.2 SO.sub.4) and evaporated. Flash
chromatography (CH.sub.2 Cl.sub.2 EtOAc 95:5) gave 0.14 g (54%)
(2S,4R)-4-[2-(2,5-Difluoro-4-methoxy-benzyloxymethyl)-4-tritylsulfanyl-pyr
rolidin-1-yl]-2-methoxy-pyrimidine, MS: 640 (MH.sup.+).
TFA/triisopropylsilane deprotection, (see General method for
S-deprotection, Method D) gave
(3R,5S)-5-(2,5-Difluoro-4-methoxy-benzyloxymethyl)-1-(2-methoxy-pyrimidin-
4-yl)-pyrrolidine-3-thiol, MS: 398 (MH.sup.+).
In analogy: a)
2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine and 10 eq phenol/NaH after 8 h at
70.degree. C. gave
2-Phenoxy-4-[(2S,4R)-2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-
pyrrolidin-1-yl]-pyrimidine, MS: 690 (MH.sup.+), which was
deprotected (see General method for S-deprotection, Method D) to
give
(3R,5S)-1-(2-Phenoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-p
yrrolidine-3-thiol, MS: 448 (MH.sup.+).
3.2 Reaction in Other Solvents
A solution of 0.24 (0.4 mmol)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine in 1 ml MeOH was treated at 0.degree. C.
with 0.16 ml (0.88 mmol) sodium methylate (5.5 M in MeOH) and kept
at this temperature (2 h), warmed up and heated for 10 h at
75.degree. C. After evaporation and extraction with aqueous
saturated NH.sub.4 Cl/Et.sub.2 O (3.times.), the organic phases
were washed with aq. 10% NaCl, dried (Na.sub.2 SO.sub.4) and
evaporated to give 0.19 g (77%)
2-Methoxy-4-[(2S,4R)-2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-
pyrrolidin-1-yl]-pyrimidine, MS: 628 (MH.sup.+), which was
deprotected (see General method for S-deprotection, Method D) to
give
(3R,5S)-1-(2-Methoxy-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-p
yrrolidine-3-thiol, MS: 386 (MH.sup.+).
In analogy: a)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine and 2.2 eq sodium methanethiolate/2 eq
sodium iodide in THF (28 h at 70.degree. C.) gave after
deprotection (see General method for S-deprotection, Method D)
(3R,5S)-1-(2-Methylsulfanyl-pyrimidin-4-yl)-5-(2,4,5trifluoro-benzyloxymet
hyl)-pyrrolidine-3-thiol, MS: 402 (MH.sup.+); b)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine and 10 eq aniline/3.5 eq
N-ethyldiisopropylamine in dioxane (48 h at 105.degree. C.) gave
after deprotection (see General method for S-deprotection, Method
D)
(3R,5S)-1-(2-Phenylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol trifluoro-acetate (1:1), MS: 447 (MH.sup.+);
c)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine and 7.5 eq benzylamine/3.5 eq
N-ethyldiisopropylamine in dioxane (48 h at 90.degree. C.) gave
after deprotection (see General method for S-deprotection, Method
D)
(3R,5S)-1-(2-Benzylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; trifluoro-acetate (1:1), MS: 461
(MH.sup.+); d)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine and 7.5 eq butylamine/3.5 eq
N-ethyldiisopropylamine in dioxane (48 h at 90.degree. C.) gave
after deprotection (see General method for S-deprotection, Method
D)
(3R,5S)-1-(2-Butylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethyl
)-pyrrolidine-3-thiol; trifluoro-acetate (1:1), MS: 427 (MH.sup.+);
e)
(2S,4R)-2-Chloro-4-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine and 30 eq methylamine solution (8.03 M
in EtOH)/3.5 eq N-ethyldiisopropylamine in dioxane (48 h at
90.degree. C.) gave after deprotection (see General method for
S-deprotection, Method D)
(3R,5S)-1-(2-Methylamino-pyrimidin-4-yl)-5-(2,4,5-trifluoro-benzyloxymethy
l)-pyrrolidine-3-thiol; trifluoro-acetate (1:1), MS: 385
(MH.sup.+).
Example 4
Suzuki-type Reactions
In general the reactions were carried out according to Stanforth,
Stephen P. Catalytic cross-coupling reactions in biaryl synthesis.
Tetrahedron (1998), 54(3/4), 263-303.
4.1 Method A (The Solvents Were Degased for 10 Min with Argon).
A solution of 1.35 g (2 mmol)
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine in 12 ml dimethoxyethane were added to a
suspension of 0.116 g (0.1 mmol)
tetrakis(triphenylphosphine)palladium in 1.4 ml dimethoxyethane and
stirred for 15 min. 0.29 g (2.4 mmol) Phenylboronic acid in 3.4 ml
EtOH was then added and after 10 min, 8.8 ml of a aqueous 2 M
Na.sub.2 CO.sub.3 solution. The reaction was heated for 2 h at
90.degree. C., evaporated and extracted wit H.sub.2 O/Et.sub.2 O
(3.times.). The organic phases were washed with aqueous 10% NaCl,
dried (Na.sub.2 SO.sub.4) and evaporated. Purification by
flash-chromatography on silica gel (toluene) gave 0.48 g (36%)
(2S,4R)-5-Phenyl-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-p
yrrolidin-1-yl]-pyrimidine, MS: 674 (MH.sup.+).
TFA/triisopropylsilane deprotection (see General method for
S-deprotection, Method D) gave
(3R,5S)-1-(5-Phenyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol, MS: 432 (MH.sup.+).
4.2 Method B (The Solvents Were Degassed for 10 Min with Argon);
Parallel Synthesis:
A solution of 0.13 mmol
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine, 0.195 mmol boronic acid or boronic acid
ester and 0.004 mmol PdCl.sub.2 (dppf) in 2 ml dioxane and 0.4 ml 2
M Na.sub.2 CO.sub.3 were heated for 48 h at 80.degree. C. After
filtration the mixture was purified by preparative HPLC (RP18, 50%
to 95% Acetonitrile).
TFA/triisopropylsilane deprotection (see General method for
S-deprotection, Method D) gave the free thiol.
4.3 Method C: (The Solvents Were Degased for 10 Min with Argon)
[Giroux, Andre; Han, Yongxin; Prasit, Petpiboon. One pot biaryl
synthesis via in situ boronate formation. Tetrahedron Lett. (1997),
38(22), 3841-3844]:
A solution of 0.68 g (1 mmol)
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine, 0.28 g (1.1 mmol)
bis(pinacolato)diboron, 0.29 g (3 mmol, dried 2 h at 100.degree.
C., 0.1 Torr) potassium acetate and 0.024 g (0.03 mmol) PdCl.sub.2
(dppf) in 12 ml DMF were stirred for 4.5 h at 80.degree. C. The
reaction was cooled, treated with 0.195 ml (2 mmol)
2-bromopyridine, 0.024 g (0.03 mmol) PdCl.sub.2 (dppf) and 2.5 ml
aqueous 2 M Na.sub.2 CO.sub.3 solution and heated for 16 h at
80.degree. C. The reaction evaporated (60.degree. C./0.1 torr) and
partitioned between water/Et.sub.2 O (3.times.). The organic phases
were washed with aqueous 10% NaCl and dried over Na.sub.2 SO.sub.4.
Purification by flash-chromatography on silica gel (toluene/EtOAc
97.5:2.5) gave 0.125 g
(2S,4R)-5-Pyridin-2-yl-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulf
anyl-pyrrolidin-1-yl]-pyrimidine, MS: 675 (MH.sup.+).
TFA/triisopropylsilane deprotection (see General method for
S-deprotection, Method D) gave
(3R,5S)-1-(5-Pyridin-2-yl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymeth
yl)-pyrrolidine-3-thiol; compound with trifluoro-acetic acid, MS:
433 (MH.sup.+).
4.4 Method D: (The Solvents Were Degased for 10 Min with
Argon):
A solution of 0.68 g (1 mmol)
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine, 15 mg (0.022 mmol) of
bis(triphenylphosphine)palladium(II)dichloride and 9.5 mg
copper(I)iodide in 0.4 ml DMF was treated at 80.degree. C. for 1 h
with a solution of 0.35 ml (2.5 mmol) ethinyltrimethylsilane and
1.87 ml Et.sub.3 N in 1.5 ml DMF. The same solution was added again
during 1 h and after 4 h extracted with pentane (3.times.)/H.sub.2
O (2.times.). The organic phase was dried (Na.sub.2 SO.sub.4),
evaporated and purified by flash-chromatography on silica gel
(toluene) to give 0.063 g (9%)
(2S,4R)-2-[2-(2,4,5-Trifluoro-benzyloxymethyl)-4-tritylsulfanyl-pyrrolidin
-1-yl]-5-trimethylsilanylethynyl-pyrimidine, MS: 694 (MH.sup.+).
TFA/triisopropylsilane deprotection (see General method for
S-deprotection, Method D) gave
(3R,5S)-5-(2,4,5-Trifluoro-benzyloxymethyl)-1-(5-trimethylsilanylethynyl-p
yrimidin-2-yl)-pyrrolidine-3-thiol, MS: 452 (MH.sup.+).
According to an analogous method the following compounds were
prepared via reaction of
(2S,4R)-5-Bromo-2-[2-(2,4,5-trifluoro-benzyloxymethyl)-4-tritylsulfanyl-py
rrolidin-1-yl]-pyrimidine with the 2.educt mentioned in the
following table 2.
TABLE 2 By the reaction of
(2S,4R)-5-Bromo-2-(2-(2,4,5-trifluoro-benzyloxymethyl)-4-
tritylsulfanyl-pyrrolidin-1-yl)-pyrimidine 68-5011 with the 2.educt
following method B. NAME 2. Educt MS COLOR
(3R,5S)-1-(5-Pyridin-4-yl-pyrimidin- 4-PYRIDYLBO- 433 M+H+ orange
2-yl)-5-(2,4,5-trifluoro- RONIC ACID
benzyloxymethyl)-pyrrolidine-3-thiol (3R,5S)-1-(5-Thiophen-3-yl-
THIOPHENE-3- 438 M+H+ pyrimidin-2-yl)-5-(2,4,5-trifluoro- BORONIC
ACID benzyloxymethyl)-pyrrolidine-3-thiol
(3R,5S)-1-[5-(4-Methoxy-phenyl)- 4-METHOXY- 462 M+H+
pyrimidin-2-yl]-5-(2,4,5-trifluoro- BENZENEBO- benzyloxy
methyl)-pyrrolidine-3-thiol RONIC ACID
(2S,4R)-4-{2-[4-Mercapto-2-(2,4,5- 4-CARBOXY- 476 M+H+
trifluoro-benzyloxy methyl)- BENZENEBO-
pyrrolidin-1-yl]-pyrimidin-5-yl}- RONIC ACID benzoic acid
(3R,5S)-1-(5-Allyl-pyrimidin-2-yl)-5- 2-ALLYL-4,4,5,5- 396 M+H+
(2,4,5-trifluoro-benzyloxy methyl)- TETRAMETHYL-
pyrrolidine-3-thiol 1,3,2-DIOXA- BOROLAN
(3R,5S)-1-(5-Pyridin-3-yl-pyrimidin- PYRIDINE-3- 433 M+H+
2-yl)-5-(2,4,5-trifluoro- BORONIC ACID
benzyloxymethyl)-pyrrolidine-3-thiol 1,3-PROPANEDIOL
Example 5
S-Acetyl-Derivatization
A solution of 397 mg (1 mmol)
(3R,5S)-1-(5-Propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-py
rrolidine-3-thiol (the trifluoro-acetate salt was extracted with
aqueous saturated NaHCO.sub.3 /EtOAc) in 6 ml pyridine were treated
at 0.degree. C. with 0.14 ml (2 mmol) acetyl chloride and stirred
for 5 h at RT. The reaction was poured on ice water and extracted
wit Et.sub.2 O (3.times.). The organic phases were washed with
aqueous 1 N HCl and 10% NaCl, dried (Na.sub.2 SO.sub.4) and
evaporated. Flash chromatography on silica gel (CH.sub.2 Cl.sub.2
/Et.sub.2 O 100:0 to 95:5) gave 383 mg (87%) (3R,5S)-Thioacetic
acid
S-[1-(5-propyl-pyrimidin-2-yl)-5-(2,4,5-trifluoro-benzyloxymethyl)-pyrroli
din-3-yl]ester, MS: 440 (MH.sup.+).
Example 6
Amines
a) To 25.0 g (52.56 mmol)
(2S,4R)-2-Hydroxymethyl-4-tritylsulfanyl-pyrrolidine-1-carboxylic
acid tert-butyl ester in 80 ml CH.sub.2 Cl.sub.2 were added 40 ml
TFA at 0.degree. C., and the solution was stirred at RT over night.
The solution was concentrated in vacuo, and the residue was
redissolved in EtOAc, washed with sat. NaHCO.sub.3 solution, brine,
and was dried over Na.sub.2 SO.sub.4. 21.98 g (quant.)
(2S,4R)-(4-Tritylsulfanyl-pyrrolidin-2-yl)-methanol were isolated
as light brown foam.
b) The crude product was suspended in 16.46 g (105.1 mmol, 2 eq)
2-chloro-5-n-propylpyrimidine and 30 ml (175 mmol, 3.3 eq)) N-ethyl
diisopropylamine and the mixture was heated to 80.degree. C. When
everything was dissolved, 350 mg (1.84 mmol) copper iodide were
added, and the reaction mixture was kept at 80.degree. C. over
night. After cooling to RT, the mixture was diluted with
EtOAc/H.sub.2 O, and the aqueous solution was extracted with EtOAc.
The combined organic layers were washed with 1M KHSO.sub.4, 1M HCl,
and brine, and were dried with Na.sub.2 SO.sub.4. Purification with
flash chromatography with EtOAc:hexane (1:4 to 1:1) yielded 19.1 g
(74%)
(2S,4R)-[1-(5-Propyl-pyrimidin-2-yl)-4--tritylsulfanyl-pyrrolidin-2-yl]-me
thanol as light yellow foam, MS: 496 (MH.sup.+).
c) 1.0 g (2.0 mmol)
(2S,4R)-[1-(5-Propyl-pyrimidin-2-yl)-4-tritylsulfanyl-pyrrolidin-2-yl]-met
hanol in 15 ml THF were treated with 764 mg (2.82 mmol) triphenyl
phosphine and 420 mg (2.82 mmol) phthalimide at RT. The solution
was cooled to 0.degree. C. and 615 .mu.l (3.83 mmol) diethylazo
dicarboxylate in 3 ml THF were added. The solution was stirred at
RT over night, H.sub.2 O was added and the inorganic layer was
extracted with ETOAc. The combined layers were washed with 1M NaOH,
sat. NaHCO.sub.3 solution and brine, and were dried over Na.sub.2
SO.sub.4. Column chromatography with EtOAc:hexane 1:2 as eluent
yielded 1.20 g (95%)
(2S,4R)-2-[1-(5-Propyl-pyrimidin-2-yl)-4-tritylsulfanyl-pyrrolidin-2-ylmet
hyl]-isoindole-1,3-dione as white solid, MS: 625 (MH.sup.+).
d) 960 mg (1.52 mmol)
(2S,4R)-2-[1-(5-Propyl-pyrimidin-2-yl)-4-tritylsulfanyl-pyrrolidin-2-ylmet
hyl]-isoindole-1,3-dione in 95 ml ethanol were treated with 2.4 ml
(49.4 mmol) hydrazine hydrate at reflux. After cooling to RT, the
solution was filtered and concentrated, the crude product was
purified by flash chromatography with CH.sub.2 Cl.sub.2
:MeOH:NH.sub.4 OH 90:10:0.25 yielding 659 mg (88%)
(2S,4R)-C-[1-(5-Propyl-pyrimidin-2-yl)-4-tritylsulfanyl-pyrrolidin-2-yl]-m
ethylamine as white foam, MS: 495 (MH.sup.+).
e) To 643 mg (1.3 mmol)
(2S,4R)-C-[1-(5-Propyl-pyrimidin-2-yl)-4-tritylsulfanyl-pyrrolidin-2-yl]-m
ethylamine in 3 ml methanol were added 158 .mu.l (1.43 mmol)
2,5-difluorobenzaldehyde and 5 ml methanol to partially redissolve
the compound. This was followed by a solution of 108 mg (0.78 mmol)
zinc chloride and 109 mg (1.56 mmol) NaBH.sub.3 CN in 3 ml
methanol. The solution was stirred over night, concentrated and
dissolved in ETOAc/sat NaHCO.sub.3 solution. The inorganic layer
was extracted with EtOAc, the combined organic layers were washed
with NaHCO.sub.3 and brine, dried over Na.sub.2 SO.sub.4 and
evaporated. Purification with column chromatography yielded 750 mg
(93%)
(2S,4R)-(2,5-Difluoro-benzyl)-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsulfany
l-pyrrolidin-2-ylmethyl]-amine as light yellow gum, MS: 621
(MH.sup.+).
f) 160 mg (0.258 mmol)
(2S,4R)-(2,5-Difluoro-benzyl)-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsulfany
l-pyrrolidin-2-ylmethyl]-amine in 2 ml pyridine were treated with
37 .mu.l (0.52 mmol) acetyl chloride at 0.degree. C. The solution
was stirred at RT for 1.5 h, poured on ice water and was extracted
with EtOAc. The combined organic layers were washed with 1N HCl and
brine, dried over Na.sub.2 SO.sub.4 and were evaporated. Column
chromatography with EtOAc:hexane 1:2 to 1:1 yielded 170 mg (quant)
(2S,4R)-N-(2,5-Difluoro-benzyl)-N-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsul
fanyl-pyrrolidin-2-ylmethyl]-acetamide as white foam.
g) From
(2S,4R)-N-(2,5-Difluoro-benzyl)-N-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsul
fanyl-pyrrolidin-2-ylmethyl]-acetamide was prepared analogously to
general procedure E
(2S,4R)-N-(2,5-Difluoro-benzyl)-N-[4-mercapto-1-(5-propyl-pyrimidin-2-yl)-
pyrrolidin-2-ylmethyl]-acetamide as colorless gum, MS: 421
(MH.sup.+).
h) From
(2S,4R)-(2,5-Difluoro-benzyl)-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsulfany
l-pyrrolidin-2-ylmethyl]-amine was prepared analogously to general
procedure E
(2S,4R)-5-[(2,5-Difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-
pyrrolidine-3-thiol as colorless gum, MS: 379 (MH.sup.+).
i) 50 mg (0.13 mmol)
(2S,4R)-5-[(2,5-Difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-
pyrrolidine-3-thiol in 1 ml pyridine were treated with 28 .mu.l
(0.39 mmol) acetyl chloride at 0.degree. C. The solution was
stirred at RT for 1.5 h, poured on ice water and was extracted with
EtOAc. The combined organic layers were washed with 1N HCl and
brine, dried over Na.sub.2 SO.sub.4 and were evaporated. Column
chromatography with EtOAc:hexane 1:1 to 2:1 yielded 54 mg (88%)
(3R,5S)-Thioacetic acid
S-[5-[[acetyl-(2,5-difluoro-benzyl)-amino]-methyl]-1-(5-propyl-pyrimidin-2
-yl)-pyrrolidin-3-yl]ester as off-white gum, Ms: 463
(MH.sup.+).
j) 220 mg (0.35 mmol)
(2S,4R)-(2,5-Difluoro-benzyl)-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsulfany
l-pyrrolidin-2-ylmethyl]-amine in 6.5 ml CH.sub.2 Cl.sub.2 were
treated with 74 .mu.l (0.43 mmol) N-ethyl diisopropylamine, 63
.mu.l (0.43 mmol) chloro benzyl formate and 26.6 mg (0.043 mmol)
DMAP polymer bound at 0.degree. C. for 5 min, and 1 h at RT. 212 mg
(0.212 mmol) polymer bound trisamine were added and the solution
was shaken over night. Filtration and concentration yielded 331 mg
(quant)
(2S,4R)-(2,5-Difluoro-benzyl)-[1-(5-propyl-pyrimidin-2-yl)-4-tritylsulfany
l-pyrrolidin-2-ylmethyl]-carbamic acid benzyl ester as white foam,
which were treated according to procedure E to give:
(2S,4R)-(2,5-Difluoro-benzyl)-[4-mercapto-1-(5-propyl-pyrimidin-2-yl)-pyrr
olidin-2-ylmethyl]-carbamic acid benzyl ester as colorless foam,
MS: 513 (MH.sup.+).
k) From
(2S,4R)-(2,5-Difluoro-benzyl)-[4-mercapto-1-(5-propyl-pyrimidin-2-yl)-pyrr
olidin-2-ylmethyl]-carbamic acid benzyl ester was prepared
analogously to example 6 g (3R,5S)-Thioacetic acid
S-[5-[[benzyloxycarbonyl-(2,5-difluoro-benzyl)-amino]-methyl]-1-(5-propyl-
pyrimidin-2-yl)-pyrrolidin-3-yl]ester.
l) To 137 mg (2.5 mmol) (3R,5S)-Thioacetic acid
S-[5-[[benzyloxycarbonyl-(2,5-difluoro-benzyl)-amino]-methyl]-1-(5-propyl-
pyrimidin-2-yl)-pyrrolidin-3-yl]ester in 5 ml EE were added 320
.mu.l 33% HBr in acetic acid at 0.degree. C. The solution was
stirred at RT for 12 h, poured on NaHCO.sub.3 and the inorganic
phase was extracted with EtOAc. The combined organic layers were
washed with brine, dried over Na.sub.2 SO.sub.4 and were
evaporated. Purification with column chromatography with EtOAc as
eluent yielded 65 mg (63%) (3R,5S)-Thioacetic acid
S-[5-[(2,5-difluoro-benzylamino)-methyl]-1-(5-propyl-pyrimidin-2-yl)-pyrro
lidin-3-yl]ester as colorless oil, MS: 421(MH.sup.+). (Nicht in
Liste).
Example A
Tablets containing the following ingredients can be manufactured in
a conventional manner:
Ingredients Per tablet Compound of formula I 10.0-100.0 mg Lactose
125.0 mg Maize starch 75.0 mg Talc 4.0 mg Magnesium stearate 1.0
mg
Example B
Capsules containing the following ingredients can be manufactured
in a conventional manner:
Ingredients Per capsule Compound of formula I 25.0 mg Lactose 150.0
mg Maize starch 20.0 mg Talc 5.0 mg
Example C
Injection solutions can have the following composition:
Compound of formula I 3.0 mg Gelatine 150.0 mg Phenol 4.7 mg Water
for injection solutions ad 1.0 ml
Example D
500 mg of compound of formula I are suspended in 3.5 ml of Myglyol
812 and 0.08 g of benzyl alcohol. This suspension is filled into a
container having a dosage valve. 5.0 g of Freon 12 under pressure
are filled into the container through the valve. The Freon is
dissolved in the Myglyol-benzyl alcohol mixture by shaking. This
spray container contains about 100 single dosages which can be
applied individually.
* * * * *